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Creutzfeldt-Jakob disease (CJD) is a fatal degenerative neurological disease. Progressive dementia is its chief clinical sign, and its characteristic pathological feature is the formation of vacuoles within the degenerating brain. These vacuoles give the brain a 'spongy' appearance, and hence CJD and some similar diseases have been termed spongiform encephalopathies. Similar diseases include Kuru in man and scrapie in sheep. These diseases are often included among the so-called slow virus diseases, but the nomenclature is misleading, since the pathogenic agents responsible for the spongiform encephalopathies are unlike any known virus. In addition, Creutzfeldt-Jakob disease is unique among the spongiform encephalopathies. Unlike Kuru and scrapie, which have clearly infectious causes in nature, CJD occurs sporadically, and the majority of cases cannot be traced to any infectious transmission. Nevertheless, brain tissue from patients with CJD can be used to transmit a similar neurological disease to experimental animals. In most parts of the world, CJD affects primarily gray matter, that part of the brain where nerve cells and their interconnections predominate. In Japan, however, roughly 38 percent of cases affect the white matter of the brain as well; white matter consists of regions of the brain where long fibers insulated by their white myelin sheaths predominate. Since this form of CJD affects most areas of the brain, it has been termed 'panencephalopathic'. Japanese researchers have now shown that it is possible to isolate a pathogenic agent from panencephalopathic CJD and use it to transmit disease to laboratory animals. However, the disease caused in guinea pigs and hamsters affects specific areas of gray matter, and is not of the panencephalopathic type. There may be an interaction between the genetic characteristics of the pathogenic agent and its human host, and the panencephalopathic disease seen so commonly in Japan is likely to result from the influence of an unknown human gene or genes that occur more frequently in Japanese populations. (Consumer Summary produced by Reliance Medical Information, Inc.)

We established a clonal cell line, HAT.MC8, derived from a human pulmonary large cell carcinoma with sarcomatoid features. This cell line was successfully maintained in a protein-free medium and exhibited sarcomatoid fibroblastic features in vitro. The cells constitutively produced a large amount of interleukin 6 (IL-6) in vitro. Tumour necrosis factor alpha (TNF-alpha) not only stimulated HAT.MC8 cells to produce IL-6, but also induced a morphological change from sarcomatoid fibroblastic to epithelial features. Although this change was related to actin and zonula adherens, there was no evidence that E-cadherin participated in the change. Interleukin 1 beta (IL-1 beta) had a stimulatory effect on IL-6 production by HAT.MC8 cells, but no influence on the morphology of the cells. [ABSTRACT FROM AUTHOR]

To identify a novel regulatory factor involved in brain development or synaptic plasticity, we applied the differential display PCR method to mRNA samples from NMDA-stimulated and un-stimulated neocortical cultures. Among 64 cDNA clones isolated, eight clones were novel genes and one of them encodes a novel zinc-finger protein, HIT-4, which is 317 amino acid residues (36-38 kDa) in length and contains seven C2H2 zinc-finger motifs. Rat HIT-4 cDNA exhibits strong homology to human ZNF597 (57% amino acid identity and 72% homology) and identity to rat ZNF597 at the carboxyl region. Furthermore, genomic alignment of HIT-4 cDNA indicates that the alternative use of distinct promoters and exons produces HIT-4 and ZNF597 mRNAs. Northern blotting revealed that HIT-4 mRNA (approximately 6 kb) is expressed in various tissues such as the lung, heart, and liver, but enriched in the brain, while ZNF597 mRNA (approximately 1.5 kb) is found only in the testis. To evaluate biological roles of HIT-4/ZNF597, targeted mutagenesis of this gene was performed in mice. Homozygous (-/-) mutation was embryonic lethal, ceasing embryonic organization before cardiogenesis at embryonic day 7.5. Heterozygous (+/-) mice were able to survive but showing cell degeneration and vacuolization of the striatum, cingulate cortex, and their surrounding white matter. These results reveal novel biological and pathological roles of HIT-4 in brain development and/or maintenance.

Ten glioma cell lines were examined for abnormalities of exon 1beta of the p14 gene and then for abnormalities of the entire p14 gene with the use of previous findings of other exons. Abnormalities of exon 1beta and the entire p14 gene were detected in eight of ten cases: homozygous deletion of the entire gene in six cases, hemizygous deletion of exon 1beta with homozygous deletion of downstream exons in one case, and hemizygous deletion of the entire coding region with a missense mutation (A97V) at the C-terminal nucleolar localization domain in one case. The remaining two cases revealed no such abnormalities. p14 gene expression was observed in the latter two cases and one case with A97V mutation in the hemizygously deleted coding region, but not in the others, including one case with only exon 1beta. In the three cases with p14 gene expression, immunocytochemistry revealed p14 nucleolar staining, suggesting the retention of the functional activity of p14 protein and, in the case with the A97V mutation, an insufficient mutational effect as well. The present findings of the frequent and variable p14 gene abnormalities, including rare-type ones with or without sufficient mutational effect in glioma cell lines, might be of value for better understanding of the p14 gene and its related pathways in glioma carcinogenesis.

Nuclear factor (NF)-kappaB is known to control cellular proliferation and apoptosis. In malignant astrocytoma cells, it was reported that NF-kappaB was activated aberrantly and promoted their proliferation. Thus, inhibition of NF-kappaB activity is considered to be a promising therapeutic strategy for malignant astrocytoma. Recently, curcumin, the major constituent of turmeric, was reported to inhibit NF-kappaB activity. In this study, we investigated inhibitory effects of curcumin on NF-kappaB activity and cellular proliferation, and induction of apoptosis by curcumin in human malignant astrocytoma cell lines. Alteration of NF-kappaB activity in NP-2 human malignant astrocytoma cell line after treatment with curcumin was examined using electrophoretic mobility shift assay. Alterations of DNA synthesis and cellular growth in five human malignant astrocytoma cell lines after treatment with curcumin were examined using [(3)H]thymidine incorporation assay and the trypan blue dye exclusion method, respectively. Induction of apoptosis by curcumin in NP-2 and NP-3 human malignant astrocytoma cell lines was examined by DNA-fragmentation analysis and morphological observation. We found that the NF-kappaB activity in NP-2 was significantly reduced by curcumin. The DNA synthesis and the cellular growth were inhibited by curcumin in dose-dependent manner in all the five malignant astrocytoma cell lines. Nuclear condensation and fragmentation, and DNA fragmentation were observed in both NP-2 and NP-3 after the treatment with curcumin. These results indicate that curcumin inhibits the cellular proliferation and induces apoptosis in human malignant astrocytoma cell lines. These results are considered to be resulted from the inhibition of NF-kappaB activity by curcumin.

Ten primary central nervous system lymphomas (PCNSL, brain lymphomas) were examined for p14 gene exon 1beta deletion, mutation and methylation by Southern blot analysis, nucleotide analysis of polymerase chain reaction clones and Southern blot-based methylation assay. In Southern blot analysis, from the signal densities of the hybridized bands and their similarities to those of exons 2 and 3 in our previous quantitative study, we found that exon 1beta was homozygously deleted in four cases, hemizygously deleted in five cases and not deleted in one case. Thus, the same deletion patterns covered the entire p14 gene for all cases except for one case, which suggested the hemizygous deletion of exons 1beta and 2 and homozygous deletion of exon 3. In addition, although exon 1beta mutation is rare in various tumors, we detected a missense mutation (L50R) in one case with a hemizygous deletion. Methylation of the 5'CpG island of the p14 gene was not suggested for any case without homozygous deletion. Our observation of frequent p14 gene abnormalities (90%) and inactivation (40-60%) was in striking contrast to the same pathological subtype of systemic lymphoma in which p14 gene abnormalities and inactivation were infrequent, suggesting a difference in carcinogenesis between PCNSL and systemic lymphoma.

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).

Using reverse transcription-polymerase chain reaction (RT-PCR), six primary brain lymphomas, pathologically diagnosed as diffuse large B-cell lymphoma, were examined for rearranged VH-D-JH sequences of the immunoglobulin heavy chain gene, focusing on somatic mutations and intraclonal heterogeneity. The reliability of the isolated PCR clones was confirmed by in situ hybridization (ISH) with complementarity-determining region (CDR) 3 oligonucleotide probes. Sequence analysis of the PCR clones revealed a high frequency of somatic mutation, ranging from 8.8 to 27.3% (mean 18.2%) in the VH gene segments in all the lymphomas. A significantly lower frequency of replacement (R) mutations than expected was also seen in their frameworks (FRs) in all cases. These findings suggested that the precursor cells were germinal center (GC)-related cells in these lymphomas. However, despite extensive cloning experiments, intraclonal heterogeneity was not detected in any case except for one in which it could not be ruled out. Thus, it seemed likely that all of our brain lymphomas were derived from GC-related cells and that at least most of them were from post-GC cells.

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.

In weaver mutant mice, substitution of an amino acid residue in the pore region of GIRK2, a subtype of the G-protein-coupled inwardly rectifying K+ channel, changes the properties of the homomeric channel to produce a lethal depolarized state in cerebellar granule cells and dopaminergic neurons in substantia nigra. Degeneration of these types of neurons causes strong ataxia and Parkinsonian phenomena in the mutant mice, respectively. On the other hand, the mutant gene is also expressed in various other brain regions, in which the mutant may have effects on neuronal survival. Among these regions, we focused on the pontine nuclei, the origin of the pontocerebellar mossy fibres, projecting mainly into the central region of the cerebellar cortex. The results of histological analysis showed that by P9 the number of neurons in the nuclei was reduced in the mutant to about one half and by P18 to one third of those in the wild type, whereas until P7 the number were about the same in wild-type and weaver mutant mice. Three-dimensional reconstruction of the nuclei showed a marked reduction in volume and shape of the mutant nuclei, correlating well with the decrease in neuronal number. In addition, DiI (a lipophilic tracer dye) tracing experiments revealed retraction of pontocerebellar mossy fibres from the cerebellar cortex after P5. From these results, we conclude that projecting neurons in the pontine nuclei, as well as cerebellar granule cells and dopaminergic neurons in substantia nigra, strongly degenerate in weaver mutant mice, resulting in elimination of pontocerebellar mossy fibres during cerebellar development.

Object: It has been suggested that nuclear factor (NF)-kappaB, a pleiotropic transcription factor, controls cell proliferation. The authors examined NF-kappaB activity and its participation in the growth of human malignant astrocytoma., Methods: The authors examined NF-kappaB activity in human malignant astrocytoma cell lines and high-grade astrocytoma tissues by using electrophoretic mobility shift assays and immunohistochemical studies, respectively. In addition, messenger (m)RNA expression of p50 and RelA, which are representative subunits of NF-kappaB, and IkappaBalpha, which is a representative inhibitory protein of NF-KB, were analyzed using Northern blot hybridization in the astrocytoma cell lines. Furthermore, alterations in DNA synthesis and cell growth in the astrocytoma cell lines were examined after inhibition of NF-kappaB activity by RelA antisense oligodeoxynucleotide. The authors found NF-kappaB activity in all astrocytoma cell lines and high-grade astrocytoma tissues that were examined, but not in the fetal astrocyte strain or in normal cerebral tissue. Expression of p50, RelA, and IkappaBalpha mRNA was found in the fetal astrocyte strain and normal adult brain tissue, in addition to the astrocytoma cell lines. The relative levels of expression of these mRNAs were similar among these cell lines, the cell strain, and normal tissue. The RelA antisense oligodeoxynucleotide specifically reduced the levels of RelA mRNA expression and NF-kappaB activity in the astrocytoma cell lines, thus significantly inhibiting their DNA synthesis and cell growth., Conclusions: Human malignant astrocytoma cells have aberrant NF-KB activity, which promotes their growth. This activity is not associated with aberrant expression of p50 and RelA.

14-3-3 protein is a brain-specific protein discovered by Moore and Perez, but at present is thought to be a multifunctional protein. To clarify the brain-specific function of the protein, we intend constructing a 14-3-3 eta gene knock-out mouse. As the first step of this process, we isolated the mouse 14-3-3 eta chain gene and determined its structure. The mouse gene is about 10 kb long and composed of two exons separated by a long intron. The transcription start site was identified and the polyadenylation signals (AATAAA) were found in exon 2 of the mouse gene. In the 5'-upstream sequence, we found several cis elements including a CRE sequence, a TATA box-like sequence, and a C/EBP element. Furthermore, the distribution of 14-3-3 eta mRNA in the mouse brain was examined by in situ hybridization histochemistry. The highest signals were found in the Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and the olfactory bulb neurons of the adult mouse. Neuronal expression of 14-3-3 eta in these regions mRNA may generally increase during postnatal brain development. The distribution of protein kinase C gamma in the mouse brain was also examined by immunohistochemistry. From the distribution of 14-3-3 eta mRNA and protein kinase C gamma in the mouse brain, the involvement of these compounds in the induction and maintenance of LTP was discussed.

To investigate the effects of adenosine on endogenous Xenopus oocyte receptors, we analysed defolliculated oocytes injected with mRNAs for the G protein-activated inwardly rectifying K(+) (GIRK) channels. In oocytes injected with mRNAs for either GIRK1/GIRK2 or GIRK1/GIRK4 subunits, application of adenosine or ATP reversibly induced inward K(+) currents, although ATP was less potent than adenosine. The responses were attenuated by caffeine, a non-selective adenosine receptor antagonist. Furthermore, in uninjected oocytes from the same donor, adenosine produced no significant current. The endogenous receptor was activated by two selective A(1) adenosine receptor agonists, N(6)-cyclopentyladenosine (CPA) and N(6)-cyclohexyladenosine (CHA), and antagonized by a selective A(1) adenosine receptor antagonist, 1,3-dipropyl-8-cyclopenylxanthine (DPCPX) at moderate nanomolar concentrations, but insensitive to micromolar concentrations of selective A(2A) and A(3) adenosine receptor agonists, 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) and N(6)-(3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine (IB-MECA), respectively. However, the pharmacological characteristics of the receptor were different from those of the cloned Xenopus A(1) adenosine receptor and previously proposed adenosine receptors. The adenosine-induced GIRK currents were abolished by injection of pertussis toxin and CPA inhibited forskolin-stimulated cyclic AMP accumulation. We conclude that an adenosine receptor on the Xenopus oocyte membrane can activate GIRK channels and inhibit adenylyl cyclase via G(i/o) proteins. Moreover, our results suggest the existence of an endogenous adenosine receptor with the unique pharmacological characteristics. As the receptor was activated by nanomolar concentrations of adenosine, which is a normal constituent of extracellular fluid, the receptor may be involved in some effects through the G(i/o) protein signalling pathways in ovarian physiology.

It is well known that there are individual differences in a sensitivity to analgesics. Several lines of evidence have suggested that the level of opioid-induced analgesia is dependent on the level of expression of the mu-opioid receptor (mu-OR). However, the molecular mechanisms underlying the diversity of the level of the opioid receptor and the opioid sensitivity among individuals remain to be elucidated. In the present study, we analyzed the opioid-receptor genes of CXBK recombinant-inbred mice, which show reduced sensitivity to opioids. Northern blotting, nucleotide sequencing, and in situ hybridization histochemical analyses demonstrated that CXBK mice possessed mu-OR mRNA with a normal coding region but an abnormally long untranslated region (UTR). In addition, the mu-OR mRNA level in CXBK mice was less than in the control mice. Next, we produced littermate mice that had inherited two copies of the wild-type mu-OR gene, had inherited two copies of the CXBK mu-OR gene, and had inherited both copies of the mu-OR genes. In these mice, inheritance of the CXBK mu-OR gene was well correlated with less mu-OR mRNA and reduced opioid effects on nociception and locomotor activity. We conclude that the CXBK mu-OR gene is responsible for the CXBK phenotypes. Because UTR differences are known to affect the level of the corresponding mRNA and protein and because UTRs are more divergent among individuals than coding regions, the present findings suggest that opioid sensitivity may vary, depending on different mu-OR levels attributable to divergent UTR of mu-OR mRNA.

With the use of RT-PCR (reverse transcriptase-polymerase chain reaction), Northern blot analysis, and Western blot analysis, seven primary brain lymphomas were examined for the state of the MMACI tumor suppressor gene. Nucleotide analysis of RT-PCR clones revealed no abnormality in the MMAC1 coding sequence in each case. Although Northern blot revealed variation among cases in the signal intensities for MMAC1 mRNA, Western blot revealed a distinct MMAC1 protein band in all cases, suggesting that the actual MMAC1 expressions were similar. In Western blot analysis of phosphorylated Akt (p-Akt), which is regulated positively by PI3K (phosphoinositide-3 kinase) and negatively by MMAC1, all the lymphomas revealed an Akt band but not a p-Akt band, suggesting that the MMAC1 phosphatase activity was maintained in each case. These findings suggest that the MMAC1 gene is normal in its coding sequence, gene expression, and phosphatase activity in the lymphomas. Thus, unlike the p16 and p15 tumor suppressor genes, which are frequently deleted and inactivated in brain lymphoma and represent a striking contrast to systemic lymphoma, MMAC1 may not play an important role in carcinogenesis in this tumor, as in the systemic counterpart.

The behavioral changes of mice induced by acute and repeated i.p. injection of phencyclidine (PCP) were observed by measuring locomotor activity and stereotyped behavior. Then, the effects of metabotropic glutamate receptor (mGluR) agonists, DCG-IV and L-CCG-1, on the above behavioral changes induced by PCP were found. The effects of DCG-IV were very strong and completely depressed the PCP-induced hyperlocomotion. The effects of L-CCG-1 were not so strong. Repeated injection of PCP for 20 days into mice induced lower locomotor activity than that in acutely injected mice. These behavioral changes may be related with the negative symptoms of schizophrenia. In order to examine some molecular mechanisms of PCP-induced behavioral changes, Northern blot analysis of total RNA from prefrontal cortical tissues of mice treated with PCP, DCG-IV, and L-CCG-1 was carried out.

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.

A cDNA clone for a novel homologue to gamma-glutamyl transpeptidase (gamma-GTP), termed GTPH, was isolated from a rat brain expression cDNA library using antisera against total brain synaptosomal fractions. The cloned GTPH consists of 641 amino acid residues (78 kDa) and exhibits structural similarity with a conventional type of gamma-GTP that is predominantly expressed in the liver: They share significant amino acid homology (33% identity, 73% similarity) spanning over the entire sequence. RNA analyses revealed that GTPH mRNA expression is found only in the nervous system, including all brain regions, eyes and peripheral ganglia, and increases during development. Endogenous GTPH protein is a membrane-bound glycoenzyme and migrates as 90-100 kDa in polyacrylamide gels. Taken together, GTPH is a novel form of a gamma-GTP-like molecule expressed exclusively in the nervous system.

A novel cDNA clone encoding a protein structurally related to the transcriptional repressor Polycomb group (PcG) proteins, which regulate homeotic genes and others, was isolated from mouse and rat brain. The coding protein contained the SPM domain and mbt repeats, both of which are characteristic of the PcG proteins, and showed significant similarity in amino acid sequence to the Drosophila Sex comb on midleg (Scm) protein. Since this novel protein contains the mbt repeats in four tandem copies, we designated this murine gene as Sfmbt for Scm-related gene containing four mbt domains. Cloning and characterization of the mouse Sfmbt gene revealed that the coding sequence comprised 20 exons, dispersed along approximately 40kb, and mapped to the proximal part of Chromosome 14. Northern blot analysis showed that the Sfmbt mRNAs were expressed most abundantly in the adult testis, and less intensively in all other tissues examined.

The expression of hydroxyindole-O-methyltransferase (HIOMT), an enzyme catalyzing the final step of melatonin biosynthesis, was examined in three pineoblastomas and five pineocytomas by in situ hybridization analysis. Distinct hybridization signals for HIOMT mRNA, though weaker than in normal pineal gland pinealocytes, were detected in two of the three pineoblastoma and three of the five pineocytoma cases. Of the pineoblastomas, hybridization signals were observed in most tumor cells of one case, while in another, signals were detected in occasional cells clustered or scattered throughout the neoplastic field. Of the pineocytomas, signals were detected in most tumor cells of two cases, while in one case, signals were detected only in occasional cells. Among these specimens, one pineoblastoma and one pineocytoma were also analyzed using northern blot and reverse transcription polymerase chain reaction (RT-PCR) analyses. In the northern blot analysis, an apparently single band corresponding to the size of HIOMT mRNA was detected in both pineoblastoma and pineocytoma RNA blots. In the RT-PCR analysis, three species of HIOMT mRNA generated via alternative splicing were detected in both tumors. These results suggest that the neoplastic cells of pineoblastomas and pineocytomas often retain the ability to express HIOMT mRNA, as in normal pinealocytes, and that HIOMT is a useful tumor marker for the diagnosis of pineal parenchymal tumors.

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.

To investigate the effects of various chemical classes of antipsychotic drugs: haloperidol, thioridazine, pimozide and clozapine, on the G-protein-activated inwardly rectifying K(+) (GIRK) channels, we carried out Xenopus oocyte functional assays with GIRK1 and GIRK2 mRNAs or GIRK1 and GIRK4 mRNAs. In oocytes co-injected with GIRK1 and GIRK2 mRNAs, application of each of the various antipsychotic drugs immediately caused a reduction of inward currents through the basally active GIRK channels. These responses were not observed in the presence of 3 mM Ba(2+), which blocks the GIRK channels. In addition, in uninjected oocytes, none of the drugs tested produced any significant current response. These results indicate that all the antipsychotic drugs tested inhibited the brain-type GIRK1/2 heteromultimeric channels. Furthermore, similar results were obtained in oocytes co-injected with GIRK1 and GIRK4 mRNAs, indicating that the antipsychotic drugs also inhibited the cardiac-type GIRK1/4 heteromultimeric channels. All the drugs tested inhibited, in a concentration-dependent manner, both types of GIRK channels with varying degrees of potency and effectiveness at micromolar concentrations. Only pimozide caused slight inhibition of these channels at nanomolar concentrations. We conclude that the various antipsychotic drugs acted as inhibitors at the brain-type and cardiac-type GIRK channels. Our results suggest that inhibition of both types of GIRK channels by these drugs underlies some of the side effects, in particular seizures and sinus tachycardia, observed in clinical practice.

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.

A total of 10 glioma cell lines were examined to evaluate the status of the MMAC1 gene, a candidate tumor suppressor gene. Six cell lines showed mutations with presumed loss of heterozygosity and 1 cell line showed no mRNA expression. The 6 mutations consisted of 3 3-bp deletions (codons 17, 101 or 199), 1 missense mutation (codon 252) and 2 truncation mutations (1 nonsense mutation at codon 233 and 12-bp insertion at codon 241). Among them, the 3-bp deletions, which are a rare type of mutation in MMAC1 gene, were located in the N-terminal half (codons 1-212) of the coding region, which is considered important in MMAC1 function. The missense mutation was located unusually in the C-terminal half (codons 212-403), but it was in a small region in which some other reported missense mutations are clustered. Thus, these 4 mutations were suggested to have functional effects on the MMAC1 activity, like the other 2 mutations with predicted protein truncations. By sequence analysis of cDNA clones, we confirmed that all the mutations including these 4 rare ones were in the MMAC1 gene, not in the PTH2 pseudogene. In 2 cases, we also examined the primary glioma tissues from which the cell lines had been derived and found the same mutations as in the cell lines in both cases. This suggested that the mutations in these cell lines were derived from the primary glioma tissues, but not from artifacts arising during long-term in vitro cultivation.

Recombinant-inbred CXBK mice have been used for various studies as putative mu-opioid-receptor deficient mice. However, CXBK mice have never been compared with gene-targeting mice lacking the mu-opioid receptor (muKO) and the K-opioid receptor (kappaKO). Here we report that CXBK mice show distinct behavioural phenotype in opioid-induced analgesia and sedation. Intraperitoneal (i.p.) administration of morphine (3 and 10 mg kg(-1)) induced significantly lower levels of analgesia in CXBK mice than in the control C57BL/6 mice, while higher doses of morphine (30 and 100 mg kg(-1)) induced marked analgesia in CXBK mice. CXBK mice also showed lower analgesia and sedation levels than did C57 mice after i.p. administration of U-50488 (10 and 30 mg kg(-1)). The partial deficiency of sensitivity to morphine and U-50488 of CXBK mice is in sharp contrast to the complete lack of sensitivity to morphine and U-50488 in muKO and kappaKO mice, respectively. Furthermore, CXBK mice showed a lower threshold for nociceptive stimuli when they were not given an opioid, suggesting that CXBK mice could have alterations in the genes related to the nociceptive threshold. These unique behavioural phenotypes of CXBK mice suggest unique genetic alterations in CXBK mice.

Recent genetic analyses have suggested a linkage between schizophrenia and the chromosomal region 22q12-q13. 14-3-3 protein, abundant in the brain, mediates interactions between diverse molecules of biological activities; its gene was recently mapped to chromosome 22q12.1-q13.1. We therefore investigated allele frequencies of a variable number of tandem repeat (VNTR) in the 5'-noncoding region of the 14-3-3 eta chain gene in controls and schizophrenics. The frequencies of the two-repeat allele were significantly higher (P < 0.05) in the schizophrenics, and particularly in those with onset before age 22 (early-onset schizophrenics, P < 0.02), than in the controls. The odds ratio was significantly increased in the early-onset schizophrenics homozygous for the two-repeat allele (OR = 3.3, 95% CI = 1.1-9.7). The 14-3-3 eta chain gene is a potential susceptibility gene for schizophrenia, and particularly for early-onset schizophrenia.

We established two glioma cell lines from two surgical specimens obtained at different times from the same patient. One (No. 9R), which was derived from the recurrent tumor (glioblastoma, grade IV), proliferated more rapidly in vitro than the other (No. 9) from the primary tumor (slightly anaplastic astrocytoma, grade II-III). No. 9R showed heterotransplantability in nude mice, whereas No. 9 did not. These findings indicate that No. 9R has a more aggressive or malignant nature than No. 9. Both cell lines showed homozygous deletion of the representative tumor suppressor p16 and p15 genes, but no p53 gene alteration. However, examination of the overall mRNA expression profile using a commercially available cDNA-spotted membrane revealed much higher expression levels of several mRNAs, at least, in No. 9R than in No. 9, although the relationship between these mRNAs and the growth potentials remained unknown. These two cell lines, derived from the same individual, with different proliferating potentials may be useful for studies on the molecular bases of glioma malignancy and progression.