A review of the microtremor horizontal-to-vertical spectral ratio (MHVSR) method.

The single-station microtremor horizontal-to-vertical spectral ratio (MHVSR) method was initially proposed to retrieve the site amplification function and its resonance frequencies produced by unconsolidated sediments overlying high-velocity bedrock. Presently, MHVSR measurements are predominantly conducted to obtain an estimate of the fundamental site frequency at sites where a strong subsurface impedance contrast exists. Of the earthquake site characterization methods presented in this special issue, the MHVSR method is the furthest behind in terms of consensus towards standardized guidelines and commercial use. The greatest challenges to an international standardization of MHVSR acquisition and analysis are (1) the what - the underlying composition of the microtremor wavefield is site-dependent, and thus, the appropriate theoretical (forward) model for inversion is still debated; and (2) the how - many factors and options are involved in the data acquisition, processing, and interpretation stages. This paper reviews briefly a historical development of the MHVSR technique and the physical basis of an MHVSR (the what). We then summarize recommendations for MHVSR acquisition and analysis (the how). Specific sections address MHVSR interpretation and uncertainty assessment.

Dienogest improves human leucocyte antigen-DR underexpression and reduces tumour necrosis factor-α production in peritoneal fluid cells from women with endometriosis.

OBJECTIVE: To determine the immunological effect of dienogest (DNG), an oral anti-endometriosis drug, on peritoneal fluid (PF) macrophages collected from women with endometriosis. Although it has been suggested that DNG has direct effects on endometriotic cells, including decreased cell proliferation and decreased anti-inflammatory cytokine production, the effects of DNG on PF cells are unclear. STUDY DESIGN: The effects of DNG on PF cells from 34 women with endometriosis and 22 women without endometriosis (controls) were investigated. Expression of human leucocyte antigen (HLA)-DR in PF macrophages, obtained from the peritoneal cavity during laparoscopic surgery, was determined by flow cytometry. HLA-DR expression was measured again after PF cells had been cultured for 72 h in a humidified atmosphere at 37 °C in 5% CO2-95% air with or without DNG. After 72 h of incubation, the concentration of pro-inflammatory tumour necrosis factor (TNF)-α in the media was measured by enzyme-linked immunosorbent assay. RESULTS: HLA-DR expression was lower in PF macrophages from women with endometriosis compared with controls. However, after DNG treatment, HLA-DR expression in PF macrophages from women with endometriosis was increased to the same level as in controls. The TNF-α concentration in the media was decreased by DNG. CONCLUSIONS: DNG can restore the antigen-presenting ability of PF macrophages by increased HLA-DR expression, and may have an anti-inflammatory effect on PF macrophages in women with endometriosis.

Distribution of amyloid burden differs between idiopathic normal pressure hydrocephalus and Alzheimer's disease.

This study aimed to elucidate the incidence and distribution of the cortical retention of Pittsburgh compound B (PIB) in patients with idiopathic normal pressure hydrocephalus (iNPH) and clarify the differences from those in patients with Alzheimer's disease (AD). Ten patients with iNPH without any clinical signs indicative of AD were enrolled in this study. Cerebral retention of PIB in positron emission tomography (PET) in iNPH patients was compared with those in seven age-matched AD patients. The CSF levels of ß-amyloid 1-42 peptide (Aß42), which inversely decrease with cerebral amyloid burden, were also measured. Three of the ten patients with iNPH showed increased cortical PIB retention. Although the mean cortical SUV ratios were similar, the distribution of PIB retention differed widely between the patients with iNPH and AD. PIB retention was limited to the high-convexity parasagittal areas in iNPH patients, whereas it spread over the frontal and parietotemporal areas in AD. The coronal images of PIB-PET were more informative than conventional transverse images in evaluating the distribution pattern of cortical PIB retention. Two iNPH patients with higher cortical PIB retention had the lowest levels of CSF Aß42, indicating that PIB retention in iNPH would not reflect a simple delay in PIB clearance but its binding to existing Aß amyloid in the brain. Our results indicate that iNPH is one of the diseases exhibiting cortical PIB retention. The characteristic distribution of PIB retention in iNPH could be useful in the differential diagnosis between iNPH and AD.

Protumoral roles of melanoma inhibitory activity 2 in oral squamous cell carcinoma.

BACKGROUND: The role of melanoma inhibitory activity 2 (MIA2) was examined in human oral squamous cell carcinoma (OSCC). METHODS: MIA2 role was examined by immunohistochemistry of human OSCCs and knockdown studies using human 3 OSCC cell lines with MIA2 expression. RESULTS: MIA2 expression was observed in 62 (66.7%) of 93 OSCCs and was associated with tumour expansion and nodal metastasis. Melanoma inhibitory activity 2 expression was inversely correlated with intratumoral infiltration of lymphocytes. Invasion and anti-apoptotic survival were reduced by MIA2 knockdown in HSC3 cells. MOLT-3 lymphocytes infiltrating the HSC3 cell layer was enhanced by MIA2 knockdown or MIA2 depletion with the antibody. In HSC3 cells, MIA2 knockdown decreased the expressions of vascular endothelial growth factor (VEGF), VEGF-C, and VEGF-D. The downregulation of VEGF-C and -D was caused by inhibition of p38 and extracellular signal-regulated kinase (ERK)1/2, respectively. Melanoma inhibitory activity 2 was co-precipitated with integrin α4 andα5 in HSC3 cells. Integrin α4 knockdown decreased p38 phosphorylation and increased apoptosis, whereas integrin α5 knockdown decreased c-Jun N-terminal kinase (JNK) phosphorylation and apoptosis. Inhibition of JNK decreased apoptosis in the HSC3 cells. CONCLUSION: These findings suggest that the roles of MIA2 might be based on the variety of the integrins and the subtypes of mitogen-activated protein kinase.

Successful treatment of monomorphic primary central nervous system post-transplantation lymphoproliferative disorder 5 years after kidney transplantation.

A 31-year-old man underwent living-related kidney transplantation in 2004 as a consequence of primary focal segmental glomerulosclerosis (FSGS). Four years after the transplantation, we confirmed nephrotic syndrome caused by recurrent FSGS. We performed plasmapheresis and low-density lipoprotein adsorption. We also combined steroid therapy with a reduction in the dose of tacrolimus and an increased dose of mycophenolate mofetil. The nephrotic syndrome improved dramatically with this combined therapeutic approach. However, 10 months after these treatments, he revisited our hospital because of altered consciousness. We detected multiple tumor masses in his brain that were ring enhanced on contrast magnetic resonance imaging. Consequently, we suspected primary central nervous system post-transplantation lymphoproliferative disorder (CNS-PTLD). We performed a craniotomy to biopsy the brain tumors. The biopsy specimen showed Epstein-Barr virus-associated diffuse large B-cell lymphoma. There is no definitive treatment for CNS-PTLD. Therefore, we treated the primary CNS-PTLD successfully with whole-brain radiation and discontinuation of immunosuppression therapy.

MicroRNA-34a upregulation during seizure-induced neuronal death.

MicroRNAs (miRNAs) are short, noncoding RNAs that function as posttranscriptional regulators of gene expression by controlling translation of mRNAs. A subset of miRNAs may be critical for the control of cell death, including the p53-regulated miRNA, miR-34a. Because seizures activate p53, and p53-deficient mice are reportedly resistant to damage caused by prolonged seizures, we investigated the role of miR-34a in seizure-induced neuronal death in vivo. Status epilepticus was induced by intra-amygdala microinjection of kainic acid in mice. This led to an early (2 h) multifold upregulation of miR-34a in the CA3 and CA1 hippocampal subfields and lower protein levels of mitogen-activated kinase kinase kinase 9, a validated miR-34a target. Immunoprecipitation of the RNA-induced silencing complex component, Argonaute-2, eluted significantly higher levels of miR-34a after seizures. Injection of mice with pifithrin-α, a putative p53 inhibitor, prevented miR-34a upregulation after seizures. Intracerebroventricular injection of antagomirs targeting miR-34a reduced hippocampal miR-34a levels and had a small modulatory effect on apoptosis-associated signaling, but did not prevent hippocampal neuronal death in models of either severe or moderate severity status epilepticus. Thus, prolonged seizures cause subfield-specific, temporally restricted upregulation of miR-34a, which may be p53 dependent, but miR-34a is probably not important for seizure-induced neuronal death in this model.

MR imaging of ventral thalamic nuclei.

BACKGROUND AND PURPOSE: The Vim and VPL are important target regions of the thalamus for DBS. Our aim was to clarify the anatomic locations of the ventral thalamic nuclei, including the Vim and VPL, on MR imaging. MATERIALS AND METHODS: Ten healthy adult volunteers underwent MR imaging by using a 1.5T whole-body scanner. The subjects included 5 men and 5 women, ranging in age from 23 to 38 years, with a mean age of 28 years. The subjects were imaged with STIR sequences (TR/TE/TI = 3200 ms/15 ms/120 ms) and DTI with a single-shot echo-planar imaging technique (TR/TE = 6000 ms/88 ms, b-value = 2000 s/mm(2)). Tractography of the CTC and spinothalamic pathway was used to identify the thalamic nuclei. Tractography of the PT was used as a reference, and the results were superimposed on the STIR image, FA map, and color-coded vector map. RESULTS: The Vim, VPL, and PT were all in close contact at the level through the ventral thalamus. The Vim was bounded laterally by the PT and medially by the IML. The VPL was bounded anteriorly by the Vim, laterally by the internal capsule, and medially by the IML. The posterior boundary of the VPL was defined by a band of low FA that divided the VPL from the pulvinar. CONCLUSIONS: The ventral thalamic nuclei can be identified on MR imaging by using reference structures such as the PT and the IML.

Microarray profile of seizure damage-refractory hippocampal CA3 in a mouse model of epileptic preconditioning.

A neuroprotected state can be acquired by preconditioning brain with a stimulus that is subthreshold for damage (tolerance). Acquisition of tolerance involves coordinate, bi-directional changes to gene expression levels and the re-programmed phenotype is determined by the preconditioning stimulus. While best studied in ischemic brain there is evidence brief seizures can confer tolerance against prolonged seizures (status epilepticus). Presently, we developed a model of epileptic preconditioning in mice and used microarrays to gain insight into the transcriptional phenotype within the target hippocampus at the time tolerance had been acquired. Epileptic tolerance was induced by an episode of non-damaging seizures in adult C57Bl/6 mice using a systemic injection of kainic acid. Neuron and DNA damage-positive cell counts 24 h after status epilepticus induced by intraamygdala microinjection of kainic acid revealed preconditioning given 24 h prior reduced CA3 neuronal death by approximately 45% compared with non-tolerant seizure mice. Microarray analysis of over 39,000 transcripts (Affymetrix 430 2.0 chip) from microdissected CA3 subfields was undertaken at the point at which tolerance was acquired. Results revealed a unique profile of small numbers of equivalently up- and down-regulated genes with biological functions that included transport and localization, ubiquitin metabolism, apoptosis and cell cycle control. Select microarray findings were validated post hoc by real-time polymerase chain reaction and Western blotting. The present study defines a paradigm for inducing epileptic preconditioning in mice and first insight into the global transcriptome of the seizure-damage refractory brain.

Multitensor tractography enables better depiction of motor pathways: initial clinical experience using diffusion-weighted MR imaging with standard b-value.

BACKGROUND AND PURPOSE: The purpose of this work was to test the feasibility of using high angular resolution diffusion imaging (HARDI)-based multitensor tractography to depict motor pathways in patients with brain tumors. MATERIALS AND METHODS: Ten patients (6 males and 4 females) with a mean age of 52 years (range, 9-77 years) were scanned using a 1.5T clinical MR unit. Single-shot echo-planar imaging was used for diffusion-weighted imaging (repetition time, 6000 ms; excitation time, 88 ms) with a diffusion-sensitizing gradient in 32 orientations and a b-value of 1000 s/mm(2). Data postprocessing was performed using both the conventional single- and multitensor methods. The depiction rate of the 5 major components of the motor pathways, that is, the lower extremity, trunk, hand, face, and tongue, was assessed. RESULTS: Motor fibers on both lesional and contralesional sides were successfully depicted by both the single-tensor and multitensor techniques. However, with the single-tensor model, the depiction of motor pathways was typically limited to the fibers of trunk areas. With the multitensor technique, at least 4 of 5 major fiber bundles arising from the primary motor cortex could be identified. CONCLUSION: HARDI-based multitensor tractography using a standard b-value (1000 s/mm(2)) can depict the fiber tracts from the face and tongue regions of the primary motor cortex.

Diffusion-weighted MR imaging in carotid angioplasty and stenting with protection by the reversed carotid arterial flow.

BACKGROUND AND PURPOSE: Distal embolism during carotid angioplasty with stent (CAS) can be protected by a flow-reversal device. Diffusion-weighted MR imaging was used to evaluate this protective procedure and perform a comparison with the control. METHODS: Cases of CAS with protection procedures were included in this study. Sixty-five men (68 procedures) and 5 women (5 procedures), with an average age of 68.8 years, having severe carotid stenosis were treated in our department between 2002 and 2004. Eleven cases were treated with the Parodi Anti-Emboli System, with which the internal carotid blood flow is reversed by simultaneous occlusion of the proximal common carotid artery and external carotid artery. Diffusion-weighted MR imaging was performed within 1-3 days after CAS. As controls, data from diffusion-weighted MR imaging in 26 patients who had diagnostic angiography were included. RESULTS: Diffusion-weighted MR imaging in diagnostic angiography showed 11.5% appearance of ischemic spots after procedures. In the Parodi Anti-Emboli System, this value was 18.2%. In the CAS group, ischemic lesions appeared only in the hemisphere ipsilateral to carotid stenosis. There were no ischemic lesions in the opposite carotid or vertebrobasilar territory. The appearance rate of new ischemic spots was not significantly different between the control group and the group of CAS with Parodi Anti-Emboli System (chi2 test, P = .6227, Fisher exact method). CONCLUSIONS: Protection results obtained with the Parodi system were excellent and comparable with conventional angiography.

Anti-p97/VCP antibodies: an autoantibody marker for a subset of primary biliary cirrhosis patients with milder disease?

We previously reported that 12.5% of primary biliary cirrhosis (PBC) sera reacted with a 95 kDa cytosol protein (p95c) that was subsequently identified as a p97/valosin-containing protein (VCP). The clinical features and course of the six anti-p97/VCP-positive PBC patients with Scheuer's stage 1 and 2 liver biopsies were monitored for an average of 15 years. This group was compared with 50 PBC patients that did not have detectable anti-VCP. Autoantibodies to a full-length recombinant p97/VCP were assayed by immunoprecipitation. All six PBC patients with anti-VCP had antibodies to the mitochondrial pyruvate dehydrogenase complex-E2 antigen as measured by an addressable laser bead immunoassay. The first was a male with no evidence of liver failure that died of cerebral infarction at the age of 85. The second was a 73-year-old female with Hashimoto's thyroiditis who has remained clinically stable without ursodeoxycolic acid (UDCA) treatment. Although the third had no HCV antibodies, he developed hepatocellular carcinoma at the age of 76 and died of renal failure at 78. The fourth was a 50-year-old female who remained clinically stable during follow-up and the fifth with Hashimoto's thyroiditis and stable liver function following UCDA treatment. The sixth was a male patient presenting a mild clinical course. The clinical course of these patients was in contrast to the 50 comparison group PBC patients who did not have anti-p97/VCP. As the six PBC patients with anti-p97/VCP antibodies had slowly progressive liver disease and no mortality related to autoimmune liver disease, our observations suggest that this autoantibody might be an indicator of a favourable prognosis.

Elevated expression of C10orf3 (chromosome 10 open reading frame 3) is involved in the growth of human colon tumor.

After analysing gene-expression profiles of colon cancers on a cDNA microarray containing cDNAs corresponding to 23 040 human genes, we focused on a gene annotated as C10orf3 (chromosome 10 open reading frame 3), whose expression was elevated in colorectal cancers (CRC) as well as in tumors arising in the stomach, lung, pancreas, and breast. The gene encodes a putative 464-amino-acid protein containing a domain known as AAA (ATPases associated with a variety of cellular activities). Western blot analysis using an antibody to the gene product confirmed that the protein was overexpressed in nine of the 15 clinical cancer tissues examined, compared to corresponding noncancerous epithelial cells. A subsequent proteomics analysis revealed that C10orf3 product associated with the product of tumor susceptibility gene 101 (TSG101), and that C10orf3 downregulated TSG101 in a post-transcriptional manner. Expression of short interfering RNA in cells derived from CRC caused significant decreases in C10orf3 expression and inhibited growth of the transfected cells, which was associated with increased apoptotic cells. These data suggest that elevated C10orf3 expression might play an essential role in the growth of cancer cells, and that suppression of C10orf3-mediated signal transduction may be a novel therapeutic strategy to a wide range of human tumors.

Autoantibodies from primary biliary cirrhosis patients with anti-p95c antibodies bind to recombinant p97/VCP and inhibit in vitro nuclear envelope assembly.

We have reported previously that p95c, a novel 95-kDa cytosolic protein, was the target of autoantibodies in sera of patients with autoimmune hepatic diseases. We studied 30 sera that were shown previously to immunoprecipitate a 95 kDa protein from [(35)S]-methionine-labelled HeLa lysates and had a specific precipitin band in immunodiffusion. Thirteen sera were available to test the ability of p95c antibodies to inhibit nuclear envelope assembly in an in vitro assay in which confocal fluorescence microscopy was also used to identify the stages at which nuclear assembly was inhibited. The percentage inhibition of nuclear envelope assembly of the 13 sera ranged from 7% to 99% and nuclear envelope assembly and the swelling of nucleus was inhibited at several stages. The percentage inhibition of nuclear assembly was correlated with the titre of anti-p95c as determined by immunodiffusion. To confirm the identity of this autoantigen, we used a full-length cDNA of the p97/valosin-containing protein (VCP) to produce a radiolabelled recombinant protein that was then used in an immunoprecipitation (IP) assay. Our study demonstrated that 12 of the 13 (93%) human sera with antibodies to p95c immunoprecipitated recombinant p97/VCP. Because p95c and p97 have similar molecular masses and cell localization, and because the majority of sera bind recombinant p97/VCP and anti-p95c antibodies inhibit nuclear assembly, this is compelling evidence that p95c and p97/VCP are identical.

Repositioning of the vertebral artery with titanium bone fixation plate for trigeminal neuralgia.

BACKGROUND: Trigeminal neuralgia is usually treated by the padding method using Teflon felt. However this can not be done in certain cases in whom a large tortuous vertebrobasilar artery compresses the fifth nerve. The transposition method using the sling may be an alternative method. But this method is not an easy procedure and requires a relatively large craniotomy. Two cases were treated by a new and simpler effective technique. CLINICAL PRESENTATION: Two cases of the trigeminal neruralgia were treated. The first case was a 71 year-old male and the second case was a 63 year-old male. The history of the medical treatments were similar and both cases had had trigeminal nerve blocks and were prescribed carbamazepin. However, the pain control was insufficient in both cases. In both cases, three dimensional computerized tomography showed the large tortuous right vertebral artery ran just behind the clivus and compressed the right trigeminal nerve. In the second case past history showed a recent hypertensive cerebellar hemorrhage. TECHNIQUE AND RESULTS: A right suboccipital craniotomy were performed in both cases. In both cases, the right vertebral artery compressed the trigeminal nerve in a rostral direction. The sling technique with nylon sutures was tried in both cases but failed during surgery. Then, the bone fixation stainless plate was cut to 10 cm in length and pre-shaped with pliers. After being shaped, the distal end of the plate was inserted between the vertebral artery and fifth nerve and the proximal end of the plate was fixed to the skull by screw. The fifth nerve was completely isolated from the artery as they were in direct contact. After surgery, the pain disappeared completely during the follow-up of one and a half year in the first case and 9 months in the second case. CONCLUSION: The plate can be bent and curved with plier to suit each individual case. This technique is easily applied even when the slings or other isolation technique is not available and appeared to achieve the mechanically stronger reposition and fixation of a very large and tortuous artery away from the trigeminal nerve.

Basic studies on the equivalent cross-relaxation rate imaging (equivalent CRI)--phantom studies.

We have studied saturation transfer in hydrophilic, cross-linked copolymer gels from irradiated polymer protons to observed water protons, using f2 (ppm) profiles of [1 - (I(infinity)/I(0))], [(I(0)/I(infinity)) - 1] or 1/T(IS)(H2O), where I(0) and I(infinity) are the longitudinal magnetization of the observed water protons before and after long-time-f2-irradiation on polymer protons, respectively, and 1/T(IS)(H2O) is the cross-relaxation rate. (A) [1 - (I(infinity)/I(0))] (magnetization transfer ratio, MTR) was used in magnetic resonance imaging (MRI) as the MTR imaging. 1/T(IS)(H2O) (cross-relaxation rate) was used in the imaging of the magnetization transfer rate constant. This method was quite time-consuming compared with MTR imaging. However, f2 (ppm) profiles of [(I(0)/I(infinity)) - 1] correlated well with corresponding profiles of 1/T(IS)(H2O), because [(I(0)/I(infinity)) - 1] is equal to 1/[T(IS)(H2O)/T1(H2O)]. These results lead us to the conclusion that [(I(0)/I(infinity)) - 1] might be applicable to cross-relaxation rate (CR)-like imaging, i.e. equivalent CRI. (B) W (%) (dry weight) profiles of [(I(0)/I(infinity)) - 1] and 1/T(IS)(H2O), obtained by near-resonance f2-irradiation, seem to indicate participation of molecular rigidity and an amount of bound water. However, those values, monitored with off-resonance f2-irradiation, seem to be independent of monomer composition and to indicate mainly participation of rigidity, i.e. W (%) of copolymer gels.

Mechanism of protection by S-(1,2-dicarboxyethyl)glutathione triester against acetaminophen-induced hepatotoxicity in rat hepatocytes.

Treatment with the triester of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) prevented the hepatotoxicity induced by acetaminophen via elevation of the glutathione (GSH) level in rat hepatocytes. This elevation of the GSH level in rat hepatocytes by DCE-GS triester was dose- and time-dependent (2.1-fold in 24 h with 0.5 mm). DCE-GS triester increased the GSH level much more effectively than GSH, DCE-GS, and DCE-GS monoester and diester. Furthermore, the activity of y-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in GSH biosynthesis, was also increased by DCE-GS triester treatment (1.4-fold in 24 h with 1.0 mm). In contrast, with a rat liver homogenate, DCE-GS increased the y-GCS activity, whereas DCE-GS triester had no effect on this activity. These results suggested that DCE-GS triester, which is transported into hepatocytes much more effectively than DCE-GS and other DCE-GS esters due to its greater lipophilicity, was hydrolyzed to DCE-GS, and then the DCE-GS produced increased the GSH level via activation of gamma-GCS in rat hepatocytes.

Effects of photoperiod and melatonin on the development of growth hormone cells and the pituitary-adrenal axis in the Djungarian hamster, Phodopus sungorus.

The development of GH cells and the pituitary-adrenal axis was morphologically examined in male Djungarian hamsters (Phodopus sungorus) exposed to short days and those kept under long days and receiving daily afternoon injections of melatonin, from the time of weaning (20 days) until 100 days of age. The postnatal increase in area of ACTH cells under long days was inhibited in short-day-exposed or melatonin-treated animals. It was suggested that a short photoperiod may suppress, via melatonin, the development of ACTH cells. GH cells were not affected by age, photoperiod or exogenous melatonin. Under long days, the zona fasciculata decreased in volume with age, while the zona reticularis increased. Such changes in the volumes of these adrenocortical zones were depressed under short days. In addition, the volumes of the zona fasciculata and zona reticularis in long-day-housed animals became respectively larger and smaller subsequent to orchidectomy and melatonin administration. These results suggest that fasciculata cells in deeper levels become progressively differentiated into reticularis cells, that short photoperiod inhibits development of both zonae, and that such an inhibition is caused mainly by the decreased secretion of androgens.

Central GABAergic innervation of the mammalian pineal gland: a light and electron microscopic immunocytochemical investigation in rodent and nonrodent species.

Light and electron microscopic immunocytochemical observations were made to demonstrate central pinealopetal fibers immunoreactive for gamma-aminobutyric acid (GABA) and synapses between their terminals and pinealocytes in the pineal gland of four rodent (Wistar-King rat; mouse; Syrian hamster, Mesocricetus auratus; Hartley strain guinea pig) and one nonrodent (tree shrew, Tupaia glis) species. GABA-immunoreactive myelinated and unmyelinated fibers and endings were found in the parenchyma of the pineal gland of all the animals examined. In the rodent species, GABAergic fibers were mainly found in the intermediate and proximal portions of the pineal gland and were nearly or entirely absent in the distal portion of the gland. Abundant GABAergic fibers were evenly distributed throughout the gland of the tree shrew. In all the animals, the habenular and posterior commissures contained abundant GABA-positive fibers, and some of them were followed to the pineal gland. GABA-positive endings made synaptic contact with pinealocytes, occasionally in mice and guinea pigs, and frequently in tree shrews; no synapses were observed in Syrian hamsters and rats. In the pineal gland of all the animals, GABA-immunoreactive cell bodies were not detected, and sympathetic fibers were not immunoreactive for GABA. These data indicate that GABAergic fibers are main pinealopetal projections from the brain. In view of the difference in the distribution of these fibers, central GABAergic innervation may play a more significant role in nonrodents than in rodents. The frequent occurrence of GABAergic synapses on pinealocytes in the tree shrew suggests that GABA released at these synapses directly controls activity of pinealocytes of this animal.

Localization of cellubrevin-related peptide, endobrevin, in the early endosome in pancreatic beta cells and its physiological function in exo-endocytosis of secretory granules.

Cellubrevins are integral membrane proteins expressed in a wide variety of tissues and usually localized in recycling vesicles. Here, we investigated the cellular localization of a cellubrevin-related peptide, endobrevin, in pancreatic (beta) cells and its implication in the exo-endocytosis of insulin and (gamma)-amino butyric acid (GABA). Immunocytochemistry showed that endobrevin is associated with tubulo-vesicular structures, which are colocalized with early endosomes labeled by early endosome antigen (EEA)-1 in insulinoma MIN6 cells. To determine the cellular localization of endobrevin, we appended the green fluorescent protein (GFP) to endobrevin and the fusion protein was introduced into MIN6 cells. The subcellular localization of GFP-endobrevin was visualized by confocal laser microscopy. Colocalization study based on the expressed GFP-endobrevin and endocytosed Texas-Red(Tx-R) labeled transferrin receptor and immunocytochemistry with anti-EEA1 antibody revealed that endobrevin was preferentially localized in the early endosome. Then, we examined the functional role of endobrevin in the exocytosis of insulin and GABA from pancreatic (beta) cells. Endobrevin overexpression increased the amount of GABA released from MIN6 cells; in contrast, it decreased the glucose-stimulated insulin release from rat islets, MIN6 and INS1-D cells to approximately 50% of the control levels. Both in vitro and in vivo binding studies showed that endobrevin binds to syntaxin 1. Finally, using the fluorescent probe FM4-64, it was revealed that endobrevin overexpression accelerates vesicle recycling. We conclude that (1) endobrevin is localized in the early endosome in pancreatic (beta) cells and (2) endobrevin plays a physiological role in the exo-endocytosis of insulin and GABA from pancreatic (beta) cells, probably via an interaction between endocytic vesicles and the endosome.