Toshiro Kumanishi

The coxsackievirus-adenovirus receptor protein as a cell adhesion molecule in the developing mouse brain

In an attempt to elucidate the molecular mechanisms underlying neuro-network formation in the developing brain, we analyzed 130 proteolytic cleavage peptides of membrane proteins purified from newborn mouse brains. We describe here the characterization of a membrane protein with an apparent molecular mass of 46 kDa, a member of the immunoglobulin superfamily of which the cDNA sequence was recently reported, encoding the mouse homologue of the human coxsackievirus and adenovirus receptor (mCAR). Western and Northern blot analyses demonstrated the abundant expression of mCAR in the mouse brain, the highest level being observed in the newborn mouse brain, and its expression was detected in embryos as early as at 10. 5 days post-coitus (dpc), but decreased rapidly after birth. On in situ hybridization, mCAR mRNA expression was observed throughout the newborn mouse brain. In primary neurons from the hippocampi of mouse embryos the expression of mCAR was observed throughout the cells including those in growth cones on immunohistochemistry. In order to determine whether or not mCAR is involved in cell adhesion, aggregation assays were carried out. C6 cells transfected with mCAR cDNA aggregated homophilically, which was inhibited by specific antibodies against the extracellular domain of mCAR. In addition to its action as a virus receptor, mCAR may function naturally as an adhesion molecule involved in neuro-network formation in the developing nervous system.

Ethanol opens G-protein-activated inwardly rectifying K + channels

Ethanol affects many functions of the brain and peripheral organs. Here we show that ethanol opens G-protein-activated, inwardly rectifying K+ (GIRK) channels, which has important implications for inhibitory regulation of neuronal excitability and heart rate. At pharmacologically relevant concentrations, ethanol activated both brain-type GIRK1/2 and cardiac-type GIRK1/4 channels without interaction with G proteins or second messengers. Moreover, weaver mutant mice, which have a missense mutation in the GIRK2 channel, showed a loss of ethanol-induced analgesia. These results suggest that the GIRK channels in the brain and heart are important target sites for ethanol.

Functional coupling of the nociceptin/orphanin FQ receptor with the G-protein-activated K+ (GIRK) channel

Nociceptin/orphanin FQ is a heptadecapeptide which was recently isolated from brains. It induces hyperalgesia, in contrast to the analgesic effects of opioid ligands, although it and its receptor structurally resemble opioid peptides and opioid receptors, respectively. To investigate the molecular mechanism underlying nociceptin/orphanin FQ actions, we performed Xenopus oocyte expression assays, in situ hybridization histochemistry and electrophysiological analyses of neurons. We found that the nociceptin/orphanin FQ receptor is functionally coupled with the G-protein-activated K+ (GIRK) channel in Xenopus oocytes, and that the receptor mRNA and GIRK1 mRNA co-exist in various neurons, including hippocampal pyramidal cells. Furthermore, we found that nociceptin/orphanin FQ induces hyperpolarizing currents via inward-rectifier K+ channels in hippocampal pyramidal cells, suggesting that the nociceptin/orphanin FQ receptor couples with the GIRK channel in this region. We conclude that the nociceptin/orphanin FQ receptor couples with the GIRK channel in various neurons, including hippocampal pyramidal cells, thereby modulating neuronal excitability.

Distribution of prepro‐nociceptin/ orphanin FQ mRNA and its receptor mRNA in developing and adult mouse central nervous systems

Nociceptin/orphanin FQ (N/OFQ) and its receptor share similarities to opioids and their receptors in terms of the molecular structure and signaling pathway, but the two systems exhibit different actions in vivo. To understand the mechanism of N/OFQ‐system actions, we examined, by in situ hybridization analysis, the distribution of preproN/OFQ and N/OFQ receptor mRNAs in the developing and adult mouse central nervous systems (CNS). In most neural regions, preproN/OFQ mRNA was mainly expressed in a small population of middle‐sized neurons. These neurons were scattered between large projection‐type neurons or within the neuropil, suggestive of interneurons. In some other nuclei (lateral septum, bed nucleus of the stria terminalis, reticular thalamic nucleus, inferior colliculus, and rostral periolivery nucleus), preproN/OFQ mRNA was expressed in a number of large projection‐type neurons. By contrast, N/OFQ receptor mRNA was evenly expressed in most neurons of the adult CNS. Considering the inhibitory actions of N/OFQ, the distinct cellular expression pattern of the N/OFQ system suggests that the release of N/OFQ from interneurons may lower neuronal and synaptic activities of neighboring neurons, leading to integration or modulation of local circuits. Furthermore, the cellular expression pattern, distinct from that of the opioid system, may provide a possible molecular/cellular basis for the different in vivo actions of N/OFQ and opioids. In embryonic stages, both preproN/OFQ and N/OFQ receptor mRNAs were highly and widely expressed in the mantle zone, suggesting the possible importance of N/OFQ signaling in CNS development. J. Comp. Neurol. 399:139–151, 1998.

Primary central nervous system lymphoma in Japan--a retrospective, co-operative study by CNS-Lymphoma Study Group in Japan.

This manuscript reports the results of the first cooperative study on primary central nervous system lymphoma (PCNSL) in Japan. Of 196 patients registered, 170 were judged as having PCNSL. No patients were immunocompromised. Of the 170 patients with PCNSL, 93 were males and 77 were females. The mean was 56.7 years. One hundred and nineteen tumors were confirmed histopathologically, and 51 were diagnosed by neuroimaging alone. All the tumors were non-Hodgkins lymphoma. According to the Working Formulation for Clinical Usage (WF), 96 out of 119 tumors were classifiable: 53 were diffuse large cell type (55.2%), 17 immunoblastic type (17.7%), 9 diffuse small cleaved type (9.4%), 6 diffuse mixed type (6.3%), 5 polymorphous type (5.2%), 5 small lymphocytic type (5.2%) and 1 small non-cleaved type (1.0%). Of 21 tumors studied immunohistochemically, 18 were B-cell type and 3 were T-cell type. Irradiated patients (144) survived significantly longer than non-irradiated patients, (median survival time, MST: 19.2 and 2.7 months, respectively; p<0.001). There was a remarkable difference in survival among patients of the intermediate lymphomas; MST (18 months) of patients with large cell lymphoma was significantly shorter than MST (over 96 months) of patients with other intermediate grade lymphomas (small cleaved and mixed) (p < 0.001) and had no significant difference from MST (9 months) of patients with high grade lymphomas. If patients were irradiated with more than 40 Gy, higher doses and different modes of irradiation brought no further survival advantage. Chemotherapy was performed in 87 of 144 irradiated patients (60.4%). No regimens were effective in prolonging survival. Of 144 irradiated patients, a complete or partial response to initial treatment was demonstrated in 91 (63.2%) and 43 patients (29.9%), respectively. Improvement in performance status was confirmed in 82 patients (57.0%). Despite a good response to initial treatments. 88 out of 144 evaluable patients have died of PCNSL (MST: 19 months). Multivariate analysis based on the Cox hazard model revealed that histology of tumor, age at onset, performance status, and radiotherapy were prognostic factors. Neither chemotherapy nor mode of surgery was a beneficial factor.

Molecular mechanisms of analgesia induced by opioids and ethanol: is the GIRK channel one of the keys?

Opioids and ethanol have been used since ancient times for pain relief. Opioid signaling is mediated by various effectors, including G protein-activated inwardly rectifying potassium (GIRK) channels, adenylyl cyclases, voltage-dependent calcium channels, phospholipase Cbeta(PLCbeta), and mitogen-activated protein kinases, although it has been unclear which effector mediates the analgesic effects of opioids. Ethanol induces a variety of physiological phenomena via various proteins, including GIRK channels rather than via membrane lipids. GIRK channel activation by either G proteins or ethanol is impaired in weaver mutant mice. The mutant mice may therefore serve as a useful animal model for studying the role of GIRK channels in vivo. Reduced analgesia by using either opioids or ethanol in weaver mutant mice suggests that GIRK channels are important effectors in both opioid- and ethanol-induced analgesia. This hypothesis is supported by similar findings in GIRK2 knockout mice. Among the various effectors coupled with opioid receptors and various targets of ethanol, GIRK channels are the only molecules whose involvement in opioid- and ethanol-induced analgesia has been demonstrated in vivo. The GIRK channel is potentially one of the key molecules in furthering the understanding of the pain control system and in developing advanced analgesics with fewer adverse effects.

Involvement of G-protein-activated inwardly rectifying K^+ (GIRK) channels in opioid-induced analgesia

To investigate the role of G-protein-activated inwardly rectifying K+ (GIRK) channels in opioid-induced analgesia, we compared the effects of opioids in wild-type and weaver mutant mice having mutant GIRK channels. In the tail-flick and hot-plate tests, weaver mutant mice displayed significantly lower analgesia after either morphine or (-)-U-50488 administration. These findings suggest that GIRK channel activation is important in the induction of analgesia by opioids.

Abrogation of apoptosis induced by DNA‐damaging agents in human bladder‐cancer cell lines with p21/WAF1/CIP1 and/or p53 gene alterations

The p53‐inducible cyclin‐dependent kinase inhibitor, p21/WAF1/CIPI (p21), plays a pivotal role in the G1 arrest or apoptosis of cells exposed to genotoxic stimuli. To determine whether p21 is a putative tumor‐suppressor gene, p21 status was investigated in 4 human bladder‐cancer cell lines of known p53 status. A p21‐gene mutation, one base‐pair insertion at codon 20 resulting in a chain‐termination change at codon 35, was observed in one cell line, HT1376, suggesting structural or functional alteration of the p21 protein. When exposed to DNA‐damaging agents, cisplatin or mitomycin C, apoptosis was induced in RT4 with the wild‐type (wt) p53/wt p21, whereas T24 with the p53 non‐sense mutation/wt p21 was resistant. Of the other 2 cell lines with the p53 mis‐sense mutation, apoptosis was induced in SCaBER with the wt p21, but HT1376 with the p21 frame‐shift mutation was fairly resistant. These findings suggest that not only p53 alteration, but also p21 alteration, is important to prevent apoptosis induced by DNA‐damaging agents. When exposed to these agents, p53 and p21 expression was increased in RT4, and not induced in T24. p53 was not induced, but p21 expression was increased in SCaBER, whereas p53 expression was increased but p21 expression was absent in HT1376. Thus, p21 expression itself may have an important role in the induction of apoptosis by DNA‐damaging agents.

Analysis of p53 gene mutations in low- and high-grade astrocytomas by polymerase chain reaction-assisted single-strand conformation polymorphism and immunohistochemistry.

Using polymerase chain reaction-assisted single-strand conformation polymorphism (PCR-SSCP) and immunohistochemical analyses, mutations in the p53 tumor suppressor gene were examined in 19 low-and high-grade gliomas. By PCR-SSCP and nucleotide analyses, p53 gene mutation was seen in 7 gliomas. Out of the 7 mutations, 3 were located at the CpG site of the previously proposed hot-spot codons 248 and 273, 2 were at codons 171 and 214 and the other 2 were in intron 5, 1 at the splice acceptor site and the other in the vicinity of the splice donor site. The latter 4 mutations have not, or only rarely, been observed in gliomas or in other tumors. However, their effect on the structural and functional alteration of the p53 protein was suggested by positive intranuclear p53 immunostaining in neoplastic cells in 3 mutations including the 1 at the splice acceptor site. In connection with glioma grading, the p53 gene mutation was shown to have occurred in both low- and high-grade gliomas, often in most of the neoplastic cells, as suggested by lack of distinct normal bands and ladders in SSCP and direct sequencing, respectively. The absence of recurrence and malignant transformation over a considerably long postoperative time in our low-grade glioma cases suggested that the p53 gene mutation might not be sufficient for the progression from low- to high-grade gliomas. The frequency of detection of mutation was 7/19(37%) by PCR-SSCP, 8/19(42%) by immunohistochemistry and 10/19(53%) by both methods. The results of PCR-SSCP and immunohistochemistry were consistent in 14 cases(73.7%), but not in 5 cases(26.3%). Thus, the use of both methods was recommended to survey the occurrence of p53 gene mutation more accurately.

Immunohistochemical analysis of tumor-infiltrating lymphocytes and major histocompatibility antigens in human gliomas and metastatic brain tumors

A subpopulation of tumor-infiltrating lymphocytes (TILs) and the expression of major histocompatibility complex (MHC) antigens of the tumor cells were examined in 14 glioma and 13 metastatic brain tumor tissues. In both tumors, most of the TILs were T lymphocytes, and both phenotypes of the cytotoxic/suppressor and helper/inducer T lymphocyte were found. On examination of MHC antigens, beta 2-microglobulin was shown intensely on tumor cells in all cases, and the monomorphic determinant of the human leukocyte antigen (HLA-DR) was shown in 10 glioma and in 5 metastatic cases. The correlation between the number of TILs and MHC antigen expression on tumor cells was equivocal as a whole in cases of both glioma and metastatic brain tumor.