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Endoplasmic reticulum-associated degradation (ERAD) is a system by which proteins accumulated in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol and degraded by the ubiquitin-proteasome pathway. HRD1 is expressed in brain neurons and acts as an ERAD ubiquitin ligase. Amyloid precursor protein (APP) is processed into amyloid-beta peptides (Abetas) that form plaque deposits in the brains of Alzheimer's disease (AD) patients. We found significantly decreased HRD1 protein levels in the cerebral cortex of AD patients. HRD1 colocalized with APP in brain neurons and interacted with APP through the proline-rich region of HRD1. HRD1 promoted APP ubiquitination and degradation, resulting in decreased generation of Abeta. Furthermore, suppression of HRD1 expression induced APP accumulation that led to increased production of Abeta associated with ER stress. Immunohistochemical analysis revealed that suppression of HRD1 expression inhibited APP aggresome formation, resulting in apoptosis. In addition, we found that the ATF6- and XBP1-induced upregulation of ERAD led to APP degradation and reduced Abeta production. These results suggest that the breakdown of HRD1-mediated ERAD causes Abeta generation and ER stress, possibly linked to AD.

Facial nerve paralysis associated with middle ear cholesteatoma, although well known, is only occasionally encountered and its clinical features and management have been evaluated in only a few studies. We studied its clinical features associated with facial paralysis. Subjects numbered 20 [15 males and 5 females] from 3-82 years old (mean: 42.6 years) with facial nerve paralysis due to middle ear cholesteatoma undergoing surgery between 1990 and 2008. After cholesteatoma lesion resection, a limited area of the fallopian canal in which facial nerve edema or redness was evident was opened, although the epineural sheath was not incised for nerve decompression. Initial paralysis was incomplete in 13 (65%). Paralysis onset was sudden in 16 (80%). The facial paralysis outcome was favorable in 14 (70%). Facial paralysis outcome was largely favorably, although poor outcomes were observed in cases with petrosal cholesteatoma and in those undergoing surgery ≥2 months after paralysis onset.

We report a rare pediatric case of congenital cholesteatoma initially manifesting as facial paralysis. A 3-year-old boy seen for acute facial palsy had developed left facial paralysis on January 4, 2008, undergoing predonisolone therapy by a local pediatrician from January 10. Upon referred on March 5, facial palsy was a severe Grade V in House-Brackman classification. Middle-ear computed tomography showed soft tissue filling the mastoid antrum through the epitympanic recess to the tympanic cavity. Endoscopic examination identified swelling of the tympanic membrane and a yellowish-white tumor of the tympanic cavity. Based on a diagnosis of congenital cholesteatoma, he underwent surgery under general anesthesia on June 27. The cholesteatoma lesion occupied the tympanic cavity, extending to the aperture of the mastoid antrum. In staged surgery, the exposed facial nerve in the tympanic cavity appeared hyperemic and adjacent to the cholesteatoma lesion. Postoperatively, facial paralysis improved incompletely remaining in Grade III in House-Brackmann classification.

Endoplasmic reticulum–associated degradation (ERAD) is a quality control mechanism in which unfolded proteins are retro-translocated to the cytosol for degradation. Our recent study showed that suppression of expression of ubiquitin ligase HRD1, which is involved in ERAD, caused amyloid precursor protein (APP) accumulation and amyloid-β (Aβ) production. Furthermore, HRD1 protein levels were significantly lower in the cerebral cortex of Alzheimer’s disease (AD) patients. To assess whether HRD1 is involved in AD pathology, we analyzed the relationship between HRD1 protein levels and Aβ production. We found that the HRD1 level was negatively correlated with the Aβ level, suggesting the possible involvement of HRD1 in Aβ generation. Keywords:: HRD1, endoplasmic reticulum–associated degradation (ERAD), amyloid-β

We report a case of hemorrhage from brainstem cavernous angioma that caused various symptoms including neurootological, gustatory and facial sensory disturbances. A man experienced nausea and numbness of his right ear. It was confirmed that the symptom were derived not from a peripheral lesion but from retrocochlear and brainstem impairment based upon the findings of the DPOAE, ABR, VEMP, caloric test, optokinetic nystagmus etc. We detected an area of retrocochlear lesion that was responsible for both the auditory and vestibular disturbances based upon detailed neurootological examination in addition to MRI findings, and showed that the impairment was improved one year later without specific treatment.

Multiple sclerosis patients sometimes complain of vertigo as one of their symptoms. We experienced a case in which vertigo was seen as the chief complaint. MRI and neurootological examinations led to the diagnosis that the vertigo was caused by recurrent multiple sclerosis lesions of the posterior cranial fossa. We then considered five earlier multiple sclerosis patients who had vertigo. Their pure tone audiogram and caloric tests were almost normal. However, neurootological examinations ETT, OKP and gravicorder showed abnormalities. Therefore, in addition to MRI T2 and T2 FLAIR image examinations, neurootological examinations are valuable for diagnosis, especially in order to detect posterior cranial fossa lesions.

Intractable oropharyngeal mucosa disease is common and its diagnosis may be delayed if the mucosal lesion is simply a symptom of systemic disease. We report two cases of pemphigus vulgaris localized in the oropharyngeal mucosa. Case 1: A 50-year-old man showed multiple oropharyngeal erosiones and was biopsied at his first visit. The histopathological diagnosis was pemphigus vulgaris and antidesmogleinauto antibody 3 based on a blood examination positive. Case 2: A 66-year-old woman originally diagnosed histopathologically with inflamed mucosa was reconsiderd for pemphigus vulgaris and checked for antidesmogleinauto antibodies 1 and 3, both found to be positive. Based on the additional immunopathological findings of the earlier rechecked specimen, the definitive diagnosis was pemphigus vulgaris. Both cases were treated by steroids. The numerical value of antidesmogleinauto antibody gradually decreased based on the recovery from symptoms. We found antidesmogleinauto antibody blood testing to be valuable for diagnosis and for evaluating therapeutic effect and symptom progress. It is also important to check for antidesmogleinauto antibody early with such intractable oropharyngeal mucosal disease patients.

家族性膜性腎症の報告は比較的稀で，これまでの報告例は少ない。今回われわれは，品胎のうち一卵性の二児がいずれも比較的低年齢で膜性腎症を発症した症例を経験した。重金属や有機溶媒への曝露は否定的で，家族歴にも膠原病や尿異常を含む腎疾患はみられなかった。腎生検組織所見は光顕で特徴的な糸球体基底膜の肥厚とPAM染色でのスパイク形成がみられたが，電顕で大小さまざまな高電子密度沈着物が基底膜上皮下，基底膜内，内皮下，傍メサンギウム領域にみられ，二次性膜性腎症を示唆する所見であった。

The interaction of glucocorticoid modulatory element-binding protein 1 (GMEB1) with procaspase-2, -8, or -9 prevents caspase oligomerization and maturation. In the present study, we examined the effect of GMEB1 on neuronal apoptosis induced by hypoxia and oxidative stress. GMEB1 effectively attenuated caspase activation and apoptosis caused by these stresses in human neuroblastoma SK-N-MC cells, indicating that it functions as a potent inhibitor of caspase activation and apoptosis in response to oxidative stress. We propose that GMEB1 blocks pro-apoptosis signals induced by a variety of stresses.

The deterioration in the circumstances of the economy and human resources for hospitals and medical industry is now broadly recognized. We succeeded in reducing the assessed loss of the Otolaryngology Department Outpatient Clinic, which resulted in a gain of over 75% profit through the cooperation of the doctors and office staff. It was assumed that the communication and knowledge management between doctors and office staff was important. While this project was planned by a doctor, it was found that creative suggestions and ideas from office staff as professionals also had a desirable effect. It appears that the resolution of problems by establishing procedures of good communication and knowledge management leads to better quality of medical treatment and would ultimately raise awareness, resulting in a better hospital and more desirable workplace.

Human ubiquitin ligase HRD1 is involved in endoplasmic reticulum-associated degradation (ERAD). We recently reported that HRD1 interacts with Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of Parkin, and promotes Pael-R degradation, resulting in the protection of cells from the lethal accumulation of Pael-R. However, except for RING-finger domain that is necessary for ubiquitin ligase activity, the roles of other human HRD1 domains are unclear. Here, we show that the proline-rich domain of HRD1 is necessary to promote the degradation of Pael-R and that the protein’s transmembrane domain is necessary to transfer Pael-R from the endoplasmic reticulum (ER) to the cytosol. A HRD1 mutant defective in the transmembrane domain is markedly more unstable than wild-type HRD1. These results suggest that HRD1 has diverse functions besides ubiquitin ligase activity. Keywords:: HRD1, endoplasmic reticulum (ER)-associated degradation (ERAD), ubiquitin ligase, proteasome, ER stress

Brainstem disorders are often characterized by abnormal eye movements, however, the type of abnormal movements vary depending on the localization and severity of the damage. Here, we report a case of pontine hemorrhage who exhibited horizontal conjugate gaze palsy and disorder of speech discrimination. The 34-year-old man presented with eye pain and dull feeling malaise?, disorder of speech articulation and torpor paralysis ?? of the left side of the body of sudden onset, that developed while he was driving a car. Clinical examination revealed horizontal conjugate gaze palsy, both rightward and leftward, while vertical gaze and convergence were normal. Although the pure-tone audiogram and DPOAE were within normal range, ABR showed prolongation of latency, and reduction of speech discrimination was recognized. MRI revealed evidence of hematoma and edema extending from the mid-dorsal aspect to the right side of the pons. Based on the findings, the patient was diagnosed to have pontine hemorrhage due to hypertension affecting the right facial nerve nucleus, right pyramidal tract, bilateral abducens nuclei, MLF, PPRF and the auditory neural pathway. Although the symptoms of pyramidal tract and auditory disorder resolved by day 19 when he was?? moved to another hospital, the other neurological symptoms were persistent.

HRD1 is an E3 ubiquitin ligase and plays an important role in endoplasmic reticulum-associated degradation (ERAD). Parkin-associated endothelin receptor-like receptor (Pael-R) is a substrate of the E3 ubiquitin ligase parkin, which has been implicated in ER stress-induced cell death in dopamine neurons in autosomal recessive juvenile parkinsonism (AR-JP). Recently, we demonstrated that endogenous HRD1 interacts with Pael-R, and that HRD1 promotes the degradation of Pael-R and protects cell death caused by the accumulation of Pael-R. Another group recently reported that HRD1 suppresses the toxicity of polyglutamine-expanded huntingtin. However, the topographical localization of HRD1 protein in the brain, especially related to neurodegenerative disease, is unclear. In this study, we used immunohistochemistry to investigate the topographical localization of HRD1 in the brain and demonstrated that HRD1 immunoreactivity was expressed widely in the substantia nigra pars compacta (SNC) containing dopaminergic neurons and was expressed in the cerebral cortex, hippocampus, dentate gyrus, striatum, globus pallidus, and Purkinje cells of the cerebellar cortex. Furthermore, HRD1 immunoreactivity was detected in the neuronal cells but not in the glial cells. These results suggest that HRD1 may play an important role in maintaining higher brain function, including motor function or learning and memory. In addition, HRD1 may have substrates other than Pael-R that are implicated in neurodegenerative disorders.

We report a case of Hunt syndrome with the primary complaint of lower cranial nerve paralysis. The patient experienced not only lower cranial nerve paralysis, but also, finally, paralysis of the facial nerve. The patient was a 68-year-old woman. She came to the hospital complaining of herpetic vesicles around the right ear and pain, vertigo, and hoarseness, but there was no facial palsy. Hydrocortisore sodium succinate and acyclovir were administered intravenously.Twelve days after, she developed right facial palsy. Prednisolone and Valaciclovir hydrochloride were orally administered. Fifty Four days after disease onset, the function of all cranial nerves recovered completely.A review of the Japanese literature revealed 44 cases of Hunt syndrome with multiple cranial nerve palarisis from 1954-2005. In our case, the development of right facial palsy occurred later than all other symptoms. The late-onset facial palsy was only 5 cases and rare.

The feelings of dizziness and unsteadiness of the patients with fibromyalgia supposed specifically amplified by the hypersensitivity mechanism of CSS (central sensitivity syndrome) of them. The severity of subjective pain and physical distress according to the questionnaires were not correlated with the objective body sway on the stabilometer.Fibromyalgia manifests primarily as chronic pain of the entire body, but is also often associated with a variety of physical symptoms including dizziness and unsteadiness. This study assessed whether objective measures of body sway and unsteadiness of them are associated with their subjective dizziness findings.Subjects were 24 patients diagnosed with fibromyalgia, but one patient who had the past history of sudden deafness was excluded. The 23 patients were assessed by a stabilometer as the objective measures of body sway, and JFIQ (Japanese version of the fibromyalgia impact questionnaire), DHI (dizziness handicap inventory) and ABC (activities-specific balance confidence) as the subjective questionnaires.The significant correlations were shown between the scores of JFIQ and DHI, JFIQ and ABC, and DHI and ABC. Then, the body sway index of stabilometer environmental area was significantly correlated with DHI score. However, the stabilometer index was not correlated neither with JFIQ or ABC.

Background Impaired epithelial barrier function renders the airway vulnerable to environmental triggers associated with the pathogenesis of bronchial asthma. We investigated the influence of protocadherin-1 (PCDH1), a susceptibility gene for bronchial hyperresponsiveness, on airway epithelial barrier function. Methods We applied transepithelial electric resistance and dextran permeability testing to evaluate the barrier function of cultured airway epithelial cells. We studied PCDH1 function by siRNA-mediated knockdown and analyzed nasal or bronchial tissues from 16 patients with chronic rhinosinusitis (CRS) and nine patients with bronchial asthma for PCDH1 expression. Results PCDH1 was upregulated with the development of epithelial barrier function in cultured airway epithelial cells. Immunocytochemical analysis revealed that PCDH localized to cell-cell contact sites and colocalized with E-cadherin at the apical site of airway epithelial cells. PCDH1 gene knockdown disrupted both tight and adhesion junctions. Immunohistochemical analysis revealed strong PCDH1 expression in nasal and bronchial epithelial cells; however, expression decreased in inflamed tissues sampled from patients with CRS or bronchial asthma. Dexamethasone (Dex) increased the barrier function of airway epithelial cells and increased PCDH1 expression. PCDH1 gene knockdown eradicated the effect of Dex on barrier function. Conclusion These results suggest that PCDH1 is important for airway function as a physical barrier, and its dysfunction is involved in the pathogenesis of allergic airway inflammation. We also suggest that glucocorticoids promotes epithelial barrier integrity by inducing PCDH1.

Leptin is an important circulating signal for the regulation of food intake and body weight. In the present study, we investigated the effect of 2-aminopurine (2-AP), an inhibitor of double-strand RNA-activated protein kinase (PKR), on leptin signal transduction. 2-AP dose-dependently inhibited the leptin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) in HEK293 cells stably transfected with the Ob-Rb leptin receptor. On the other hand, we observed only slight inhibition of leptin-induced STAT3 activation by purine treatment, indicating that the inhibitory effect will be dramatically enhanced in the presence of an amino group. 2-AP did not inhibit PMA-induced ERK activation, indicating that the effect may be leptin-signal specific. The inhibitory effect of 2-AP was not mediated by newly synthesized protein because the inhibitory effect of 2-AP on leptin-induced STAT3 activation was not abrogated in the presence of the protein synthesis inhibitor cycloheximide. Interestingly, leptin did not induce PKR activation, suggesting that the effect of 2-AP on the leptin signal may be independent of PKR. Finally, 2-AP inhibited leptin-induced phosphorylation of the Ob-Rb leptin receptor. These results provide evidence of a novel action of 2-AP, i.e., inhibition of the activation of leptin signal transduction at the level of the Ob-Rb leptin receptor.

It has been suggested that impulsive behavior is caused by dysfunctional serotonergic 5-HT neurotransmission in the central nervous system (CNS). Brain neuroimaging studies have shown that behavioral inhibition is linked to the activation of cortex sites such as the ventral frontal cortex. Positron emission tomography (PET) imaging with [(18)F]altanserin to characterize 5-HT(2A) receptor binding revealed a reduction in 5-HT(2A) binding in the ventral frontal cortex in women who had recovered from impulsive diseases. These clinical, neuroimaging, and pharmacological studies appear to support the hypothesis that functional alteration of neurotransmission due to genetic polymorphisms of the 5-HT receptors may be involved in impulsive behavior modulation. Following evaluation by a self-reporting measure, it was proposed that a polymorphism in the promoter of the 5-HT(2A) receptor gene is the underlying cause of impulsive behavior; however, this hypothesis is not convincing. We examined whether the polymorphism in the 5-HT(2A) receptor gene promoter is involved in impulsive aggression by evaluating a behavioral task (Go/No-go task) in normal volunteers. The polymorphism of the 5-HT(2A) receptor gene promoter in lymphocytes from 71 volunteers was analyzed by using PCR. Impulsivity was defined as the number of commission errors (responding when one should not) recorded during a Go/No-go task; a larger number of commission errors indicate greater difficulty in inhibiting impulsive behavior. The subjects of the A-1438A allele group for the 5-HT(2A) receptor gene made more commission errors under the punishment-reward (PR)condition in a Go/No-go task than those in the G-1438G group. In the present review, we discuss and suggest the possible involvement of the A-1438A polymorphism of the 5HT2A receptor gene promoter in impulsive behavior. This hypothesis was evaluated by using a behavioral task measure that could directly reveal impulsive behavioral traits in humans.

Caspases are divided into two classes: initiator caspases, which include caspase-8 and - 9 and possess long prodomains, and effector caspases, which include caspase-3 and - 7 and possess short prodomains. Recently, we demonstrated that glucocorticoid modulatory element-binding protein 1( GMEB1) interacts with the prodomain of procaspase-2, thereby disrupting its autoactivation and the induction of apoptosis. Here we show that GMEB1 is also capable of binding to procaspase-8 and - 9. GMEB1 attenuated the Fas-mediated activation of these caspases and the subsequent apoptosis. The knockdown of endogenous GMEB1 using RNA interference revealed that cells with decreased GMEB1 expression are more sensitive to stress and undergo accelerated apoptosis. Transgenic mice expressing a neurospecific GMEB1 had smaller cerebral infarcts and less brain swelling than wildtype mice in response to transient focal ischemia. These results suggest that GMEB1 is an endogenous regulator that selectively binds to initiator procaspases and inhibits caspase-induced apoptosis

The aim of this study was to elucidate the role of SUMO-1 modification in caspase-7 in neuronal cells. We have previously demonstrated that procaspase-2 could be a possible target for SUMO-1 modification. In the present study, we attempted to investigate whether other caspases also interact with Ubc9/SUMO-1. The specific binding of SUMO-1 with caspase-7 was observed in mammalian cells. Deletion mutant analysis revealed that a SUMO-1 modification site may be located in at least N-terminal p20 subunit of caspase-7. Furthermore, SUMO-1-modified caspase-7 appeared as a dot-like structure in nuclear localization. These findings suggest that SUMO-1 modification in caspase-7 may be linked to specific its localization in the nucleus and may therefore contribute to the cleavage of nuclear substrates during neuronal apoptosis.

In the present study, we examined how the cell survival signaling via cyclic AMP-responsive element binding protein (CREB) and Akt, and the cell death signaling via cystein proteases, calpain and caspase-3, are involved in oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/reoxygenation)-induced cell death in nerve growth factor (NGF)-differentiated PC12 cells. OGD/reoxygenation-induced cell death was evaluated by LDH release into the culture medium. The level of LDH release was low (9.0% +/- 4.1%) immediately after 4 hr of OGD (0 hr of reoxygenation), was significantly increased to 28.6% +/- 6.6% at 3 hr of reoxygenation, and remained at similar levels at 6 and 20 hr of reoxygenation, suggesting that reoxygenation at least for 3 hr resulted in the loss of cell membrane integrity. After 4 hr of OGD followed by 3 hr of reoxygenation, dephosphorylation of phosphorylated CREB (pCREB), but not phosphorylated Akt (pAkt), was induced. Under these conditions, calpain- but not caspase-3-mediated alpha-spectrin breakdown product was increased, indicating that OGD/reoxygenation also induced an increase in calpain activity. The restoration of pCREB by protein phosphatase (PP)-1/2A inhibitors or the inhibition of excessive activation of calpain by calpain inhibitor did not reduce OGD/reoxygenation-induced LDH release. Cotreatment with PP-1/2A and calpain inhibitors reduced OGD/reoxygenation-induced LDH release. The present study suggests that a balance in the phosphorylation and proteolytic signaling is involved in the survival of NGF-differentiated PC12 cells.

Stress signals that impair the function of the endoplasmic reticulum (ER) can lead to an accumulation of unfolded proteins in the ER causing cell death. Recent studies have indicated that ER stress contributes to several diseases such as neurodegenerative disorders or diabetes. In the present study, we found that Akt down-regulation is important for inducing CHOP expression, an ER stress-induced transcription factor. Treatment with tunicamycin or thapsigargin, ER stress inducers, caused dephosphorylation of Akt from 12 to 24 h and induced cell death. Interestingly, treatment with a PI3K inhibitor alone induced CHOP expression and caused cell death. However, a MEK1 inhibitor induced neither CHOP expression nor cell death. These results indicate that the inactivation of Akt by ER stress induces CHOP expression and causes cell death. Therefore, Akt plays an important role in ER stressed condition and may have important implications for understanding ER stress-related diseases., http://www.sciencedirect.com/science/journal/0006291X

Among the 14 kinds of serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes (5-HTR), 5-HT2C receptor (5-HT2CR) has been intensively investigated because of its physiologically and pathophysiologically important role in the brain. 5-HT2CR has been suggested to be involved in depressive disorders based on findings from pharmacological/neurochemical/behavioral studies using autopsy preparations of humans suffering from depression, animal models of depression, and animals treated with antidepressant drugs. Recently the editing of 5-HT2CR mRNA has been reported to participate in the pathogenesis of depressive disease. The RNA editing of 5-HT2CR induced by the presumable alteration of deaminase during a pathological state in depression causes changes of a base to another base (e.g., adenosine to guanosine, cytidine to uracil (thymidine)), followed by changes in amino acids constituting the second intracellular transmembrane loop that couples G proteins. Thus 5-HT2CR receptor-mediated signal transduction is changed. In the present review, the pathopharmacological significance of 5-HT2CR in special reference to RNA editing of receptors is reviewed and discussed from the aspect of development of novel therapeutics for depression. Keywords:: 5-HT2C receptor, RNA editing, depression, antidepressant

The antioxidative activity and ameliorative effects on memory impairment by sulfur-containing compounds which occur in Allium vegetables such as onion and garlic were investigated. The antioxidative activities of S-alk(en)yl-L-cysteines and their sulfoxides, volatile alk(en)yl disulfides and trisulfides, and vinyldithiins were examined by using human low-density lipoprotein. It was elucidated that the alk(en)yl substituents and the number of sulfur atoms in the compounds were important for the antioxidative activities. To demonstrate the ameliorative effects on memory impairment, onion extract and synthesized di-n-propyl trisulfide were administered to senescence-accelerated mouse P8. The behavioral experiments showed that onion extract and di-n-propyl trisulfide had highly ameliorative effect of memory impairment. Furthermore, it was found that the hippocampus lipid hydroperoxide in senescence-accelerated mouse P8 was decreased by the administration of di-n-propyl trisulfide. These results suggest that di-n-propyl trisulfide contained in onion ameliorates memory impairment in SAMP8 mouse by its antioxidant effect.

We searched for a gene that is up-regulated in response to LPS at a later time point in primary cultured glial cells. Using a Gene Chip Probe Array, we identified stefin A3, which is known as a cysteine protease inhibitor. As assessed by RT-PCR, we observed a time-dependent (2 to 48 h) up-regulation of stefin A3. The results indicate that stefin A3 is involved in infection and inflammation at a later time point.

The afferent vagus nerve has been suggested to be an important component for transmitting peripheral immune signals to the brain. However, there is inconsistent evidence showing that subdiaphragmatic vagotomy did not inhibit the brain mediated behavioral and neural effects induced by the peripheral application of lipopolysaccharide (LPS). LPS triggers innate immune cells through Toll-like receptor 4 (TLR4). In the present study, we found that TLR4 mRNA and protein was expressed in the rat nodose ganglion. Thus, it is suggested that LPS could activate afferent vagus nerve at the level of nodose ganglion, which exists centrally from the subdiaphragmatic level of vagus nerve. The results could provide evidence for the novel pathway of LPS-induced afferent vagus nerve activation.

Secretory phospholipase A(2)s (sPLA(2)s) have been implicated in physiological and pathological events, but the regulatory mechanism(s) of their activities in cells remains to be solved. Previously, we reported that phenylarsine oxide (PAO), a sulfhydryl reagent, stimulated arachidonic acid (AA) release in rat pheochromocytoma PC12 cells. In this study, we examined the effects of thimerosal, another sulfhydryl reagent, to clarify the sulfhydryl modification and activation of sPLA(2) molecules in cells. Like PAO, thimerosal-stimulated AA release in an irreversible manner and the responses were not additive. Dithiol compounds such as dithiothreitol inhibited AA release from both the thimerosal- and the PAO-treated cells, and monothiol compounds (l-Cys and glutathione) decreased the thimerosal response. Both sulfhydryl reagents stimulated AA release from the HEK293T cells expressing human sPLA(2)X, and stimulated the sPLA(2) activities of bee venom sPLA(2) and the soluble fraction of sPLA(2)X-expressing cells. Our results suggest that the sPLA(2)s in cells are inactive and modification of disulfide bonds in the molecules can be a trigger of sPLA(2) activation in cells. Sulfhydryl reagents are useful tools for studying the regulatory mechanism(s) of sPLA(2) activity in cells.

Genetic analysis of learning and memory deficits (LMD) in senescence-accelerated mouse P8 (SAMP8) was performed by cross-mating SAMP8 and Japanese Fancy Mouse 1 (JF1). The incidence of LMD in the F2 generation showed a 3:1 segregation ratio of mice with LMD to normal mice, and the incidence of LMD in the backcross generation of the F1 to JF1 parental strain was in agreement with a 1:1 ratio of mice with LMD to normal mice. Estimation of the number of genes involved in the development of LMD using Wright's formula showed that at least two to four genes are involved. These results suggest that the inheritance of LMD is polygenically controlled and that there may be a single major gene, but this locus is not sex-linked. Moreover, hormonal influence on the development of LMD in SAMP8 females is of a genotype-dependent manner.

進行下咽頭癌に対する放射線化学療法の意義について検討した。比較試験の結果からはシスプラチンを主体とした化学療法を加えると根治切除術不能例では生存率延長に寄与すると考えられ, 手術可能例では喉頭温存に寄与すると考えられた。本邦の報告でも放射線化学療法を加えることによって良い生存率が得られていた。われわれの施設で行ったシスプラチンと5FUによる化学療法と放射線の併用療法では手術検体の検索で55%に局所の腫瘍が消失しており, 喉頭温存が図れることが示された。5年死因特異的および粗生存率は, それぞれ56%, 51%であった。

Polyubiquitination plays key roles in various proteasome-dependent and independent cellular events. To elucidate roles in stress response of polyubiquitin chains formed via specific chain linkages in mammalian cells, we established NIH3T3 stable cell lines that are capable of conditionally expressing K29R, K48R and K63R ubiquitin mutants, in which the Lys29, Lys48 and Lys63 residues of ubiquitin had been changed to Arg, and we examined the effects of various stresses on their cell viabilities. The expression of K63R ubiquitin mutant decreased viability of the cells post-exposed to ethanol, H(2)O(2) and methyl methanesulfonate (MMS), while that of K48R mutant decreased viability of the cells post-exposed to heat shock as well as ethanol, H(2)O(2) and MMS. Thus, these results suggest that polyubiquitin chains formed via specific chain linkages are involved in the respective stress responses in mammalian cells.

The regulatory mechanisms leading to IL-20 expression during infection have not been elucidated. In the present study, we found that bacterial lipopolysaccharide (LPS) induced IL-20 expression in the primary cultured glial cells and RAW264.7 macrophage cell line. Pretreatment with protein synthesis inhibitor puromycin or cycloheximide failed to inhibit the expression of IL-20, suggesting that the expression was not dependent on de novo protein synthesis. Myeloid differentiation factor 88 (MyD88) is an important adaptor molecule for Toll-like receptor signaling. We observed complete inhibition of LPS-induced expression of IL-20 in the primary cultured glial cells prepared from MyD88-deficient mice. Furthermore, a p38 MAP kinase inhibitor, SB203580, inhibited LPS-induced expression of IL-20 mRNA. LPS-induced p38 MAP kinase phosphorylation was delayed in MyD88-deficient glial cells. Therefore, it is suggested that LPS induces IL-20 expression through MyD88-p38-dependent mechanisms. As dexamethasone inhibited LPS-induced IL-20 expression, the expression of IL-20 is regulated by a negative feedback loop mediated through glucocorticoids. Therefore, it is suggested that IL-20 may play a crucial role in inflammatory conditions in the brain.

Neurons are highly vulnerable to ischemic/hypoxic stress, while glial cells show tolerance to such stress. However, the mechanisms for tolerance acquisition in glial cells have yet to be established. We attempted to isolate and identify a stress protein that is upregulated in response to hypoxia in human astrocytoma CCF-STTG1 cells. In particular, pyruvate kinase (PK) was upregulated by hypoxia in CCF-STTG1 cells. Hypoxia-inducible factor 1 (HIF-1), the primary transcription factor that is responsible for multiple gene activation under hypoxia, plays a critical role in PK expression during hypoxic challenge. To determine whether newly synthesized PK is involved in tolerance to hypoxic stress, we established the PK-overexpressing neuronal cells. Overexpression of the wild-type, but not the kinase-negative mutant, resulted in attenuation of the loss of cell viability and the typical apoptotic features by hypoxia or oxidative stress in SK-N-MC cells. These findings suggest that upregulation of PK may result in acquisition of tolerance against hypoxic stress, and that the antioxidant effect may be involved in the protective effect of PK.

Manganese superoxide dismutase (MnSOD) is an antioxidative enzyme that scavenges superoxide radicals and is localized in the mitochondrial matrix. MnSOD is induced by a variety of stimuli through nuclear factor (NF)-κB and AP-1 activation. We investigated the expression of MnSOD in HeLa cells exposed to various agents interfering with endoplasmic reticulum (ER) functions. All agents caused an increase in the mRNA and protein levels of MnSOD. Although ER stress-responsive genes often are up-regulated by ATF6, IRE1 and XBP1, which are ER stress-related transcription factors/transducers, the overexpression of neither molecule affected the levels of MnSOD mRNA and protein. Furthermore, we showed that ER stress reagents induced NF-κB and AP-1 activation that were inhibited by a dominant-negative IRE1 mutant. We finally demonstrated that ER stress-induced MnSOD expression was reduced by the IRE1 mutant. These results suggest that the MnSOD expression is controlled by ER stress through IRE1-mediated NF-κB and AP-1 activation., This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technalogy, Japan.

Endoplasmic reticulum (ER) stress-induced cell death plays an important role in cerebral ischemia. In the present study, we investigated whether edaravone (3-methyl-1-phenyl-pyrazolin-5-one), a free radical scavenger, can protect against ER damage induced by cerebral ischemia. In a mouse model of hypoxia/ischemia, treatment with edaravone reduced edema-corrected infarction volume, attenuated hemispheric swelling, and improved neurological status. Moreover, edaravone suppressed ER stress-mediated apoptosis by inhibiting eukaryotic initiation factor alpha phosphorylation, C/EBP homologous protein (CHOP) induction, and caspase-12 activation. In mouse primary cultured glial cells, edaravone attenuated ER stress as evidenced by inhibition of the induction of glucose regulated protein 78 and CHOP and XBP-1 splicing under treatment with tunicamycin (Tm), which induces ER stress. Tm did not induce the production of reactive oxygen species in primary cultured glial cells. In addition, the free radical scavengers N-acetyl-l-cysteine and ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] did not affect ER stress response caused by Tm. These results demonstrated a novel action of edaravone that can protect against ER dysfunction in cerebral ischemia.

The SAMP8 mouse is a suitable animal model to examine the decline in learning ability with senescence. We tried to identify genes related to brain senescence by using fluorescent differential display analysis. Total RNAs were extracted from hippocampus and cerebrum of groups of five male SAMP8 and SAMR1 (control) mice, 8 months of age. A cDNA fragment of 165 bp encoding a part of EST was cloned. An mRNA sequence of 2487 bp that consists of five exons and contains ORF (684 bp for 228 amino acids) could be linked to chromosome 15 by using EST clusters in the National Center for Biotechnology Information (NCBI) database. Expression of 2.5-kb cDNA was confirmed in hippocampus and cerebrum by RT-PCR, and sequenced. This was a novel gene, named as ankyrin small ( ank-s ), the protein of which contained 4 ankyrin-repeats in N-terminal side and membrane-binding site in C-terminus side. Northern blot showed that expression of ank-s was the highest in brain, especially in hippocampus where its expression doubled after birth and in 8-month-old SAMP8 was twice that in SAMR1 mice. These results suggest that ank-s is related to neuronal degeneration in SAMP8 hippocampus.

The senescence-accelerated mouse (SAM) P8 strain exhibits severe age-related learning and memory deficits (LMD) well before the median age of survival. As a first step to clarify the genes involved in this deterioration, we have performed genetic analysis of SAMP8 using the whole genome scan for quantitative trait loci (QTLs) specifying the impairment in step-through passive avoidance response with F2 intercrosses of SAMP8 exhibiting short retention time (RT) and Japanese Fancy Mouse 1 (JF1) derived from Japanese wild mouse ( Mus musculus molossinus ) exhibiting normal long retention time. Genetic markers were typed at 113 loci spanning all chromosomes except the Y. Five loci have been identified with significant linkage to chromosomes 1, 12, 13 and 15 by interval mapping of 264 F2 mice. Three of them on chromosomes 1, 12, and 13 are due to SAMP8 background, while two of them on chromosome 15 are derived from JF1 background despite parental JF1 strain shows normal phenotype.

Cross-mating between SAMP8 and JF1 (Japanese Fancy Mouse 1) normal mice was performed for the genetic studies. At 5 months of age, learning and memory deficits (LMD) of F2 and backcross progenies were assessed using a Step-Through passive avoidance apparatus. The F2 generation showed a 3:1 segregation ratio of LMD to normal mice, and the backcross generation segregated into a 1:1 ratio of LMD to normal mice. These results are in good agreement with Mendelian inheritance. Estimation of the number of genes using Wright's formula showed that at least two to three genes are involved in the development of LMD in SAMP8 mice. Thus, LMD in SAMP8 mice may be inherited by a relatively small number of genes and regulated by a major single gene. In further experiments, we conducted QTL analysis to identify memory dysfunction QTLs in SAMP8 mice using 264 F2 progenies. A total of 126 genetic markers were typed in all chromosomes. Four significant QTLs to LMD were identified on chromosomes 1, 12, 13, and 15. The number of these QTLs is almost consistent with the statistically estimated gene number. Congenic strains carrying the QTLs need to be produced to verify the existence of LMD genes in the candidate regions of the chromosomes.

Endoplasmic reticulum-associated degradation (ERAD) is a system in which unfolded proteins drained from the ER lumen to the cytosol are ubiquitinated then degraded by 26S proteasome. We have identified and characterized human HRD1 as a ubiquitin ligase involved in ERAD that protects against ER stress-induced cell death. Accumulation of Pael receptor (Pael-R), a substrate of Parkin, has been proposed to lead to neuronal death in Autosomal Recessive Juvenile Parkinsonism (AR-JP). HRD1 co-localized with Pael-R in the ER and interacted with Pael-R through the proline-rich region of HRD1. HRD1 ubiquitinated and degraded Pael-R through its ubiqutin ligase activity. Furthermore, we found that ATF6 and XBP1 that induce HRD1 promoted the degradation of Pael-R. A class of compounds known as chemical chaperones, such as 4-phenylbutyric acid (4-PBA), has been demonstrated to repair unfolded proteins. We demonstrated that 4-PBA protected against ER stress-induced neuronal cell death. The tunicamycin-induced up-regulation of GRP78 and GRP94 and phosphorylation of PERK was suppressed by treatment with 4-PBA, indicating that 4-PBA suppresses ER stress responses by decreasing unfolded protein. Furthermore, 4-PBA suppressed ER stress induced by the overexpression of Pael-R. Thus, up-regulation of HRD1 and 4-PBA could decrease accumulation of Pael-R.

A brief period of ischemia, i.e. preconditioning of the middle cerebral artery territory, induces ischemic tolerance reducing the cerebral infarction volume caused by subsequent lethal ischemia. Nevertheless, little is known about the molecular mechanisms underlying this phenomenon. In the present study, we examined the involvement of the activation of Akt, a serine/threonine kinase, in the cerebral ischemic tolerance. Western blot analysis showed that Akt was activated in both non-preconditioned and preconditioned groups after ischemia for 1 hr, but the activation was long-lasting in the preconditioned rats. Immunohistochemical analysis demonstrated that the preconditioning-induced preventive effect on a rapid decrease in the activation level of Akt was due to the persistent activation of Akt in the penumbra region. In addition, TUNEL staining demonstrated that the preconditioning treatment inhibited the augmentation of neuronal death probably through apoptosis in the penumbra region to prevent the spread of infarction. Since the activation of Akt has been reported to protect cells from stress, the present results suggest that the preconditioning-induced persistent activation of Akt in the penumbra region plays an important role in ischemic tolerance of the brain.

Abnormally folded proteins are susceptible to aggregation and accumulation in cells, ultimately leading to cell death. To protect cells against such dangers, expression of various genes including molecular chaperones can be induced and ER-associated protein degradation (ERAD) activated in response to the accumulation of unfolded protein in the endoplasmic reticulum (ER). This is known as the unfolded protein response (UPR). ERAD requires retrograde transport of unfolded proteins from the ER back to the cytosol via the translocon for degradation by the ubiquitin-proteasome system. Hrd1p is a UPR-induced ER membrane protein that acts as a ubiquitin ligase (E3) in the ERAD system. Hrd3p interacts with and stabilizes Hrd1p. We have isolated and identified human homologs (HRD1 and SEL1/HRD3) of Saccharomyces cerevisiae Hrd1p and Hrd3p. Human HRD1 and SEL1 were up-regulated in response to ER stress and overexpression of human IRE1 and ATF6, which are ER stress-sensor molecules in the ER. HEK293T cells overexpressing HRD1 showed resistance to ER stress-induced cell death. These results suggest that HRD1 and SEL1 are up-regulated by the UPR and contribute to protection against the ER stress-induced cell death by degrading unfolded proteins accumulated in the ER.

Two cases of nasopharyngeal cancer with intracranial extension were treated with intra-arterial cisplatin infusion with systemic infusion of sodium thiosulfate (STS) followed by continuous IV infusion of 5-FU for 120 h. After two courses of intra-arterial chemotherapy, tumors had completely disappeared. Subsequently, full doses of external irradiation were applied. Both of them survived 5 years after the initiation of the treatment, but one died of the original cancer after just 5 years.