Molecular characterization and gene disruption of a novel zinc-finger protein, HIT-4, expressed in rodent brain.

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.

Frequent and variable abnormalities in p14 tumor suppressor gene in glioma cell lines.

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.

Inhibition of cellular proliferation and induction of apoptosis by curcumin in human malignant astrocytoma cell lines.

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.

Primary malignant lymphoma of the brain: frequent abnormalities and inactivation of p14 tumor suppressor gene.

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.

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes.

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).

Primary malignant lymphoma of the brain: mutation pattern of rearranged immunoglobulin heavy chain gene.

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.

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.

Degeneration of pontine mossy fibres during cerebellar development in weaver mutant mice.

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.

Isolation and structure of the mouse 14-3-3 eta chain gene and the distribution of 14-3-3 eta mRNA in the mouse brain.

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.

Aberrant nuclear factor-kappaB activity and its participation in the growth of human malignant astrocytoma.

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.

Functional characterization of an endogenous Xenopus oocyte adenosine receptor.

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.

Immunohistochemical Changes of the Transcription Regulatory Factors in Rat Striatum after Methamphetamine Administration.

We have found evidence for cyclic adenosine monophosphate (cAMP) response element (CRE) in the 5'-upstream region of the human 14.3.3 η chain gene during studies on isolation and structure of animal brain 14.3.3 cDNA and the human 14.3.3 h chain gene. cAMP response element-binding protein (CREB) and phosphorylated CREB (pCREB) may bind to this CRE. Since it was considered that these CREBs may play an important role in molecular mechanisms in the brain of animals treated with methamphetamine, we examined the expression of CREB and pCREB in rat brain after acute and chronic methamphetamine administrations using antibodies against CREB and pCREB. We observed findings for change in the expression of these factors. Our findings should be discussed in relation to the data of other authors.