Heterogeneity and potentiation of AMPA type of glutamate receptors in rat cultured microglia

alpha-amino-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor in rat cultured microglia were analyzed precisely using flop- and flip-preferring allosteric modulators of AMPA receptors, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA) and cyclothiazide (CTZ), respectively. Glutamate (Glu)- or kainite (KA)-induced currents were completely inhibited by a specific blocker of AMPA receptor, LY300164, indicating that functional Glu-receptors in cultured microglia are mostly AMPA receptor but not KA receptor in many cells. Glu- and KA-induced currents were potentiated by PEPA and CTZ in a concentration-dependent manner. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide varied with cells between 0.1 and 0.9, suggesting cell-to-cell heterogeneity of AMPA receptor subunits expressed in microglia. Quantitative RT-PCR revealed that GluR1-3 mainly occurred in the flip forms, which agreed with the stronger potentiation of receptor currents by CTZ vs. PEPA. Finally, the potentiation of microglial AMPA receptors by PEPA and CTZ inhibited the Glu-induced release of tumor necrosis factor-alpha (TNF-alpha) unpredictably. The increase in TNF-alpha release by Glu or KA required extracellular Na+ and Ca2+ ions but not mitogen-activated protein kinase (MAPK), suggesting the effects of PEPA and CTZ were not due to the inhibition of MAPK. These results suggest that potentiation of microglial AMPA receptors serves as a negative feedback mechanism for the regulation of TNF-alpha release and may contribute to the ameliorating effects of allosteric modulators of AMPA receptors.     

Clinicopathological study of diffuse neurofibrillary tangles with calcification. With special reference to TDP-43 proteinopathy and alpha-synucleinopathy

Diffuse neurofibrillary tangles with calcification (DNTC) is a relatively rare presenile dementia that clinically shows overlapping symptoms of Alzheimer's disease and frontotemporal lobar degeneration (FTLD). DNTC is pathologically characterized by localized temporal or frontotemporal atrophy with massive neurofibrillary tangles, neuropil threads and Fahr's-type calcification without senile plaques. We tried to clarify the molecular basis of DNTC by immunohistochemically examining the appearance and distribution of accumulated alpha-synuclein (aSyn) and TAR DNA-binding protein of 43kDa (TDP-43) in the brains of 10 Japanese autopsy cases. We also investigated the clinically characteristic symptoms from the clinical charts and previous reports, and the correlations with neuropathological findings. The characteristic symptoms were evaluated using the Neuropsychiatric Inventory Questionnaire (NPI-Q). As a result, we confirmed the high frequency of neuronal cytoplasmic accumulation of aSyn (80%) and phosphorylated TDP-43 (90%) in DNTC cases. There was a significant correlation between some selected items of NPI-Q scores and the severity of the limbic TDP-43 pathology. The pathology of DNTC included TDP-43 and aSyn pathology with high frequency. These abnormal accumulations of TDP-43 might be involved in the pathological process of DNTC, having a close relationship to the FTLD-like psychiatric symptoms during the clinical course.     

Pacovatinins A-C, new labdane diterpenoids from the seeds of Renealmia exaltata

Three new labdane diterpenoids, pacovatinins A-C (1-3), were isolated from seeds of the Brazilian medicinal plant Renealmia exaltata ("Pacová-catinga"), and their structures including absolute configurations were elucidated by spectroscopic data and a modified Mosher method.     

Two closely related ubiquitin C-terminal hydrolase isozymes function as reciprocal modulators of germ cell apoptosis in cryptorchid testis

The experimentally induced cryptorchid mouse model is useful for elucidating the in vivo molecular mechanism of germ cell apoptosis. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. Here, we analyzed the function of two closely related members of the ubiquitin C-terminal hydrolase (UCH) family in testicular germ cell apoptosis experimentally induced by cryptorchidism. The two enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. The testes of gracile axonal dystrophy (gad) mice, which lack UCH-L1, were resistant to cryptorchid stress-related injury and had reduced ubiquitin levels. The level of both anti-apoptotic (Bcl-2 family and XIAP) and prosurvival (pCREB and BDNF) proteins was significantly higher in gad mice after cryptorchid stress. In contrast, Uchl3 knockout mice showed profound testicular atrophy and apoptotic germ cell loss after cryptorchid injury. Ubiquitin level was not significantly different between wild-type and Uchl3 knockout mice, whereas the levels of Nedd8 and the apoptotic proteins p53, Bax, and caspase3 were elevated in Uchl3 knockout mice. These results demonstrate that UCH-L1 and UCH-L3 function differentially to regulate the cellular levels of anti-apoptotic, prosurvival, and apoptotic proteins during testicular germ cell apoptosis.     

Cross‐sectional analysis of germline BRCA1 and BRCA2 mutations in Japanese patients suspected to have hereditary breast/ovarian cancer

The prevalence of BRCA1/2 germline mutations in Japanese patients suspected to have hereditary breast/ovarian cancer was examined by a multi‐institutional study, aiming at the clinical application of total sequencing analysis and validation of assay sensitivity in Japanese people using a cross‐sectional approach based on genetic factors estimated from personal and family histories. One hundred and thirty‐five subjects were referred to the genetic counseling clinics and enrolled in the study. Full sequencing analysis of the BRCA1/2 gene showed 28 types of deleterious mutations in 36 subjects (26.7%), including 13 types of BRCA1 mutations in 17 subjects (12.6%) and 15 types of BRCA2 mutations in 19 subjects (14.1%). Subjects were classified into five groups and 22 subgroups according to their personal and family history of breast and/or ovarian cancer, and the prevalence of deleterious mutations was compared with previously reported data in non‐Ashkenazi individuals. Statistical analysis using the Mantel‐Haenszel test for groups I through IV revealed that the prevalence of Japanese subjects was significantly higher than that of non‐Ashkenazi individuals (P = 0.005, odds ratio 1.87, 95% confidence interval 1.22–2.88). Family history of the probands suffering from breast cancer indicated risk factors for the presence of deleterious mutations of BRCA1/2 as follows: (1) families with breast cancer before age 40 within second degree relatives (P = 0.0265, odds ratio 2.833, 95% confidence interval 1.165–7.136) and (2) families with bilateral breast cancer and/or ovarian cancer within second degree relatives (P = 0.0151, odds ratio 2.88, 95% confidence interval 1.25–6.64). (Cancer Sci 2008; 99: 1967–1976)     

cDNA and genomic sequences for rat 8-oxo-dGTPase that prevents occurrence of spontaneous mutations due to oxidation of guanine nucleotides

The enzyme, 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase),is present In various organisms and plays an important rolein control of spontaneous mutagenesis. This enzyme degrades8-oxoguamne-contaning deoxyribonucleoside triphosphate, a potentiallymutagenic substrate for DNA synthesis, to the correspondingmonophosphate. To obtain appropriate probes for expression ofthe gene in various tissues and also to construct appropriateexperimental models for carcino genesis, we cloned cDNA forrat 8-oxo-dGTPase and elucidated its structure. The nucleotidesequence of the cDNA revealed that the rat 8-oxo-dGTPase proteinis composed of 156 amino acid residues. The molecular weightof rat 8-oxo-dGTPase, calculated from the predicted amino acidse was 18 006, and the 8-oxo-dGTPase protein of this size wasdetected when the cDNA was expressed in 8-oxo-dGTPase-deficlentEscherichitz coil mutT^– cells. The predicted amino acidsequence of the rat 8-oxo-dGTPase has a close homology withthose of human and bacterial counterparts. Using the cDNA asa probe, part of the rat gene for 8-oxo-dGTPase was isolatedand was found to consist of at least three exons and spannedabout 10 kb. A genomic region containing the pseudogene wasalso isolated.     

Osteoarthrosis of upper extremity--basic conception and knack of diagnosis

Joints of upper extremities are non weight-bearing joints. However, there are various diseases which cause pain in these joints with various frequencies. Elaborate movements of hand are the basis of many activities in daily life and reach function of elbow joint enables washing of faces and eating. Thus, pain in these joints hinders basic daily activities. There are only few cases that we treat as primary osteoarthrosis. Almost all cases are secondary osteoarthrosis which are caused by various trauma or diseases. It is very difficult and almost impossible to recover completely even with appropriate medical treatment once osteoarthrosis occurred. Therefore, it is important that we acknowledge those trauma and diseases which cause osteoarthrosis in these joints. It is also important to educate patients in order to prevent progression of osteoarthrosis.     

Disturbed spatial learning of rats after intraventricular administration of transforming growth factor-beta 1

Patients with subarachnoid hemorrhage (SAH) who later suffer hydrocephalus show persistently high levels of transforming growth factor-beta 1 (TGF-beta 1) in the cerebrospinal fluid after the onset of SAH. Recombinant TGF-beta 1 induces hydrocephalus in mice. This study examined the spatial learning ability of rats after intraventricular administration of TGF-beta 1. Thirteen-week-old Wistar rats were treated with 0.8 or 8.0 micrograms of human recombinant TGF-beta 1 by direct injection or via osmotic pump. Three months later, their spatial learning ability was evaluated with a Morris water maze. Ventricular size, ultrastructural features, and sodium-potassium-adenosine triphosphatase (Na+, K(+)-ATPase) activity of the subarachnoid space were examined. All three TGF-beta 1-treated groups clearly exhibited impaired spatial learning ability, but they did not exhibit ventricular dilation. Histological examination revealed subarachnoid fibrosis and deactivation of Na+, K(+)-ATPase in the arachnoid cells. These findings are similar to those of our previous experiments involving injection of TGF-beta 1 in mice. The present and previous studies suggest that subarachnoid fibrosis is an important factor in the disturbance of the spatial learning ability of rats, whereas ventricular size is less important.     

Studies on the risk factors for fenofibrate-induced elevation of liver function tests

We evaluated risk factors for elevation of liver function test values after administration of fenofibrate to 45 hyperlipidemic patients. The effects of 23 factors of physical examinations, clinical laboratory test values, and the background of the patients on the elevation of liver function test values were analyzed by the logistic regression method. The increase of the values of liver function tests was found to be correlated with BMI, serum levels of triglycerides, and ALP before therapy, especially sex, serum gamma-GTP level before therapy and reduced serum triglyceride levels. These results suggest that caution must be exercised to avoid liver dysfunction in patients treated with fenofibrate.