Histone modifications in status epilepticus induced by kainate.

Animal models of epilepsy have allowed the determination of the basic molecular and cellular mechanisms of epileptogenesis. Generalized limbic seizures and subsequent status epilepticus can be induced by either pilocarpine, the muscarinic acetylcholine receptor agonist or kainate, the glutamate receptor agonist. There has been increasing interest that chromatin remodeling might play a critical role in gene regulation even in non-dividing cells such as neurons. One form of chromatin remodeling is histone amino-terminal modification that can generate synergistic or antagonistic affinities for the interactions of transcriptional factors, in turn causing changes in gene activity. Two widely studied histone modification processes are histone acetylation and phosphorylation. While histone hyperacetylation indicates an increase in gene activity, its hypoacetylation marks gene repression. Both states are controlled by a dynamic interplay of histone acetyltransferase (HAT) and histone deacetylase (HDAC). We have found the upregulation of acetylation and phosphorylation of histones, coupled with status epilepticus after kainate administration. c-fos and c-jun mRNA have been sequentially induced in response to kainate, in different hippocampal subpopulations starting from the dentate gyrus and spreading to the cornus ammonis regions well correlated with the spatio-temporal distribution of histone H4 hyperacetylation. Both histone modifications are associated with the c-fos gene promoter after kainate stimulation, while only histone acetylation with the c-jun gene. Pretreatment with curcumin, which has a HAT inhibitory activity specific for CBP/p300, attenuates histone modifications, IEGs expression and also the severity of status epilepticus after kainate treatment. Histone modifications may have a crucial role in the development of epilepsy induced by kainate.

Possible expression of functional glutamate transporters in the rat testis.

Neither expression nor functionality is clear in peripheral tissues with the molecular machineries required for excitatory neurotransmitter signaling by L-glutamate (Glu) in the central nervous system, while a recent study has shown that several Glu receptors are functionally expressed in the rat testis. This fact prompted us to explore the possible functional expression in the rat testis of the Glu transporters usually responsible for the regulation of extracellular Glu concentrations in the brain. RT-PCR revealed the expression, in the rat testis, of mRNA for five different subtypes of Glu transporters, in addition to that for particular subtypes of ionotropic and metabotropic Glu receptors. Glutamate transporter-1 (GLT-1) was different in the brain from that in the testis in terms of molecular sizes on Northern and Western blot analyses. In situ hybridization as well as immunohistochemical analysis showed localized expression of glutamate aspartate transporter at interstitial spaces and GLT-1 at elongated spermatids in the rat testis respectively. The expression of mRNA was localized for excitatory amino acid transporter-5 at the basal compartment of the seminiferous tubule in the rat testis. [(3)H]Glu was accumulated in testicular crude mitochondrial fractions in a temperature- and sodium-dependent saturable manner with pharmacological profiles similar to those shown in brain crude mitochondrial fractions. These results suggested that particular subtypes of central Glu transporters for the regulation of extracellular Glu concentrations in the rat testis could be constitutively and functionally expressed.

Necdin acts as a transcriptional repressor that interacts with multiple guanosine clusters.

Necdin is a growth suppressor expressed predominantly in postmitotic neurons, and ectopic expression of this protein suppresses cell growth. Here we report that Necdin directly binds to specific DNA sequences and serves as a transcriptional repressor. Polyhistidine-tagged Necdin was used for selection of random-sequence oligonucleotides by polymerase chain reaction-based amplification. Necdin recognized guanosine (G)-rich sequences that encompass multiple G clusters and intervening mono- or di-nucleotides of A, T and C. These sequences, termed GN boxes, resemble multiply aligned forms of the canonical GC box which is recognized by Sp family members. Necdin directly bound to a GN box consisting of contiguous two GC boxes with four G clusters, but not to a single GC box with two G clusters, whereas Sp1 bound to both. In a reporter system using Drosophila Schneider Line 2 cells, Necdin repressed Sp1-dependent activity of mouse c-myc P1 promoter that contains a typical GN box. Deletion of the GN box from the c-myc P1 promoter or its conversion to the single GC box abolished the Necdin-dependent repression. These results suggest that Necdin modulates gene transcription via the GN box that is potentially recognized by GC box-targeting Sp family members.

Prognostic factors and survival after hepatic resection for hepatocellular carcinoma without cirrhosis.

BACKGROUND: Detailed follow-up of patients with chronic hepatitis has resulted in increased diagnosis of hepatocellular carcinoma (HCC) in patients without cirrhosis. Despite numerous studies on hepatic resection, the prognostic factors for intrahepatic recurrence and survival are not well known for patients with HCC without cirrhosis. METHODS: Among 349 patients with HCC treated in the past 13 years, cirrhosis was absent in 126 patients (36 per cent). Curative hepatic resection was carried out in 100 (79 per cent) of these patients. Risk factors for intrahepatic recurrence and prognostic factors for survival were evaluated by univariate and multivariate analyses. RESULTS: Postoperative morbidity and mortality rates were 22 and 3 per cent respectively. The 5- and 10-year disease-free and overall survival rates were 31 and 50 per cent, and 22 and 47 per cent respectively. Blood loss, surgical resection margin, intrahepatic metastasis, portal vein invasion and extent of hepatic resection were independently associated with overall survival. However, the only risk factors for intrahepatic recurrence were portal vein invasion and hepatitis C virus (HCV) infection. The former was related to early recurrence while the latter was related to later recurrence. The 5-year disease-free survival rate was 58 per cent in patients with hepatitis B virus infection while it was 6 per cent in patients with HCV infection (P < 0.001). CONCLUSION: In the treatment of HCC without cirrhosis, major hepatectomy is advocated to prevent early recurrence. Liver transplantation may be required for patients with HCV infection.

The postmitotic growth suppressor necdin interacts with a calcium-binding protein (NEFA) in neuronal cytoplasm.

Necdin, a growth suppressor expressed predominantly in postmitotic neurons, interacts with viral oncoproteins and cellular transcription factors E2F1 and p53. In search of other cellular targets of necdin, we screened cDNA libraries from neurally differentiated murine embryonal carcinoma P19 cells and adult rat brain by the yeast two-hybrid assay. We isolated cDNAs encoding partial sequences of mouse NEFA and rat nucleobindin (CALNUC), which are Ca(2+)-binding proteins possessing similar domain structures. Necdin interacted with NEFA via a domain encompassing two EF hand motifs, which had Ca(2+) binding activity as determined by (45)Ca(2+) overlay. NEFA was widely distributed in mouse organs, whereas necdin was expressed predominantly in the brain and skeletal muscle. In mouse brain in vivo, NEFA was localized in neuronal perikarya and dendrites. By immunoelectron microscopy, NEFA was localized to the cisternae of the endoplasmic reticulum and nuclear envelope in brain neurons. NEFA-green fluorescent protein (GFP) fusion protein expressed in neuroblastoma N1E-115 cells was retained in the cytoplasm and partly secreted into the culture medium. Necdin enhanced the cytoplasmic retention of NEFA-GFP and potentiated the effect of NEFA-GFP on caffeine-evoked elevation of cytosolic Ca(2+) levels. Thus, necdin and NEFA might be involved in Ca(2+) homeostasis in neuronal cytoplasm.

Characterization and chromosomal mapping of a human Necdin pseudogene.

The necdin gene is expressed predominantly in postmitotic neurons and encodes a growth suppressor that interacts with the transcription factors E2F1 and p53. Human necdin gene (NDN) is maternally imprinted and located in Prader-Willi syndrome deletion region 15q11.2-q12. We isolated an NDN homologous sequence from a human genomic DNA library. The homologous sequence is overall 83% identical with necdin cDNA sequence, and possesses a short poly(A) stretch at the 3' end and direct repeats at both ends. Expression of the homologous sequence, which lacks a 5' promoter sequence, was undetected in cultured human cell lines. We mapped this sequence to chromosome 12q14-q21.1 by fluorescence in situ hybridization. These characteristics of the NDN-homologous sequence are consistent with those of processed pseudogenes. The information about the necdin pseudogene in the human genome will be useful for genetic studies on NDN-associated neurogenic disorders.

Regulation and deregulation of E2F1 in postmitotic neurons differentiated from embryonal carcinoma P19 cells.

Neurons withdraw from the cell cycle immediately after differentiation from their proliferative precursors. E2F1, a principal transcription factor that promotes cell cycle progression, must be silenced in neurons. We investigated the E2F1 system in postmitotic neurons derived from murine embryonal carcinoma P19 cells. P19 cells highly expressed the E2F1 gene during neural differentiation, and enriched neurons contained a high abundance of E2F1 mRNA. In contrast, postmitotic neurons possessed extremely low levels of E2F1 protein as assessed by the electrophoretic mobility shift assay and Western blotting. A recombinant E2F1 fusion protein was ubiquitinated in vitro when incubated with neuronal lysates. In addition, treatment with the proteasome inhibitor MG132 increased the endogenous level of E2F1 protein in neurons. These results suggest that the ubiquitin-proteasome pathway contributes, at least in part, to the downregulation of E2F1 protein in postmitotic neurons. Adenovirus-mediated transfer of E2F1 cDNA into postmitotic neurons induced both bromodeoxyuridine incorporation and chromatin condensation, suggesting that deregulated E2F1 expression causes both aberrant S-phase entry and apoptosis of postmitotic neurons. Thus, downregulation of endogenous E2F1 protein in postmitotic neurons may be indispensable for the prevention of their reentry into the cell cycle.

Physical and functional interactions of neuronal growth suppressor necdin with p53.

Necdin is expressed in virtually all postmitotic neurons, and ectopic expression of this protein suppresses cell proliferation. Necdin, like the retinoblastoma protein, interacts with cell cycle promoting proteins such as simian virus 40 large T antigen, adenovirus E1A, and the transcription factor E2F1. Here we demonstrate that necdin interacts with the tumor suppressor protein p53 as well. The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to a narrow region (amino acids 35-62) located between the MDM2-binding site and the proline-rich region in the amino-terminal domain of p53. The electrophoretic mobility shift assay showed that necdin supershifted a complex between p53 and its binding DNA, implying that the p53-necdin complex is competent for DNA binding. In p53-deficient osteosarcoma SAOS-2 cells, necdin markedly suppressed p53-dependent activation of the p21/WAF promoter. Necdin and p53 inhibited cell growth in an additive manner as assessed by the colony formation of SAOS-2 cells, suggesting that necdin does not affect p53-mediated growth suppression. On the other hand, necdin inhibited p53-induced apoptosis of osteosarcoma U2OS cells. Thus, necdin can be a growth suppressor that targets p53 and modulates its biological functions in postmitotic neurons.

The human chromosomal gene for necdin, a neuronal growth suppressor, in the Prader-Willi syndrome deletion region.

Necdin is a growth suppressor expressed in virtually all postmitotic neurons in the brain. The human necdin gene, NDN, is maternally imprinted and deleted in the Prader-Willi syndrome, a neurobehavioral contiguous gene disorder. Here, we isolated and characterized the human chromosomal necdin gene and its promoter region. The necdin gene is intronless, and it encodes a protein of 321 amino acid residues, four residues shorter than mouse Necdin. By fluorescence in-situ hybridization analysis, the necdin gene was localized to chromosome 15q11.2-q12 within the Prader-Willi syndrome deletion region. CpG islands were found in a region extending from the proximal 5'-flanking sequence to the protein coding region. The 5'-flanking sequence, which lacks canonical TATA and CAAT boxes, possessed a promoter activity in postmitotic neurons derived from murine embryonal carcinoma P19 cells. Methylation in vitro of HhaI CpG sites in the promoter region reduced the transcriptional activity. These results suggest that the necdin gene is silenced through methylation of the CpG island encompassing its promoter region.

Necdin, a postmitotic neuron-specific growth suppressor, interacts with viral transforming proteins and cellular transcription factor E2F1.

Necdin is a nuclear protein expressed in virtually all postmitotic neurons, and ectopic expression of this protein strongly suppresses the proliferation of NIH3T3 cells. Simian virus 40 large T antigen targets both p53 and the retinoblastoma protein (Rb) for cellular transformation. By analogy with the interactions of the large T antigen with these nuclear growth suppressors, we examined the ability of necdin to bind to the large T antigen. Necdin was co-immunoprecipitated with the large T antigen from the nuclear extract of necdin cDNA-transfected COS-1 cells. Yeast two-hybrid and in vitro binding analyses revealed that necdin bound to an amino-terminal region of the large T antigen, which encompasses the Rb-binding domain. Moreover, necdin bound to adenovirus E1A, another viral oncoprotein that forms a specific complex with Rb. We then examined the ability of necdin to bind to the transcription factor E2F1, a cellular Rb-binding factor involved in cell-cycle progression. Intriguingly, necdin, like Rb, bound to a carboxyl-terminal domain of E2F1, and repressed E2F-dependent transactivation in vivo. In addition, necdin suppressed the colony formation of Rb-deficient SAOS-2 osteosarcoma cells. These results suggest that necdin is a postmitotic neuron-specific growth suppressor that is functionally similar to Rb.

Expression and functional analysis of a novel isoform of gicerin, an immunoglobulin superfamily cell adhesion molecule.

We have cloned a novel cDNA of gicerin, a cell adhesion molecule belonging to the immunoglobulin superfamily. Both gicerin isoforms share the same extracellular domain, which has five immunoglobulin-like loop structures and a transmembrane domain as s-gicerin, but differ in the cytoplasmic tail domain. As the newly identified form has a larger cytoplasmic domain than the previously reported form, we refer to them as l-gicerin and s-gicerin, respectively. l-gicerin is transcribed from a distinct mRNA containing an inserted sequence not found in s-gicerin mRNA which caused a frameshift for the coding region for a cytoplasmic domain. Previous studies demonstrated that gicerin showed a doublet band of 82 and 90 kDa in chicken gizzard smooth muscle. We report that the 82-kDa protein corresponds to s-gicerin and the 90-kDa protein to l-gicerin. We also found that the two gicerin isoforms are expressed differentially in the developing nervous system. Functional analysis of these gicerin isoforms in stable transfectants revealed that they had differ in their homophilic adhesion properties, as well as in heterophilic cell adhesion assayed with neurite outgrowth factor. In addition, these isoforms have neurite-promoting activity by their homophilic adhesion, but differ in their ability to promote neurite outgrowth.

A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones.

Interaction of chromatin with the nuclear envelope and lamina is thought to help determine higher order chromosome organization in the interphase nucleus. Previous studies have shown that nuclear lamins bind chromatin directly. Here we have localized a chromatin binding site to the carboxyl-terminal tail domains of both A- and B-type mammalian lamins, and have characterized the biochemical properties of this binding in detail. Recombinant glutathione-S-transferase fusion proteins containing the tail domains of mammalian lamins C, B1, and B2 were analyzed for their ability to associate with rat liver chromatin fragments immobilized on microtiter plate wells. We found that all three lamin tails specifically bind to chromatin with apparent KdS of 120-300 nM. By examining a series of deletion mutants, we have mapped the chromatin binding region of the lamin C tail to amino acids 396-430, a segment immediately adjacent to the rod domain. Furthermore, by analysis of chromatin subfractions, we found that core histones constitute the principal chromatin binding component for the lamin C tail. Through cooperativity, this lamin-histone interaction could be involved in specifying the high avidity attachment of chromatin to the nuclear envelope in vivo.

Repeated hepatic dearterialization for unresectable liver metastases from gastric cancer: review of five cases.

A novel method of repeated hepatic dearterialization was evaluated in five patients with multiple metastases from gastric cancer in both hepatic lobes. After gastrectomy with extensive lymph node dissection (R2/3), all patients underwent implantation of a vascular occluder around the hepatic artery. Cannulation of the hepatic artery was added for later chemotherapy. The hepatic artery was occluded repeatedly for 1 hour twice daily in combination with intrahepatic infusion of anticancer drugs for as long as possible. Three of five patients demonstrated marked tumour regression with unexpectedly long survival (16 months in two patients and one still alive at 15 months). Carcinoembryonic antigen (CEA) levels decreased to almost normal in four patients who had initially high levels. The present experiences seems to indicate that long survival can be hoped for in patients with advanced gastric cancer with unresectable liver metastases.

Expression of gicerin in development, oncogenesis and regeneration of the chick kidney.

Neurite outgrowth factor, which promotes neurite extension from neuronal cells, is an extracellular matrix glycoprotein belonging to the laminin family. Gicerin is a protein that binds neurite outgrowth factor. Its cDNA cloning has revealed that it is a novel cell adhesion molecule belonging to the immunoglobulin super-family. Functional analysis demonstrates that gicerin possesses homophilic binding activity as well as heterophilic binding activity with neurite outgrowth factor. We examined the role and expression of neurite outgrowth factor and gicerin in chick kidney during development. In the embryonic kidney, gicerin was found to be highly expressed both on ureteric bud cells and metanephrogenic mesenchymal cells, when the mesenchymal cells become condensed to be converted into polarized epithelial cells. In the adult kidney, the expression of gicerin was decreased and restricted to the glomerulus, proximal tubule and medullary loop. On the other hand, neurite outgrowth factor was constitutively expressed in the basement membranes of tubules and the matrices of glomeruli during development. As some molecules which are expressed during embryogenesis and suppressed after maturation are re-expressed in tumor cells or tissues during regeneration, we also examined the expression of gicerin in chicken Wilms' tumor and regenerating kidney in interstitial nephritis. Gicerin was remarkably upregulated in Wilms' tumor and re-expressed in collecting ducts recovering from interstitial nephritis. These findings suggest that gicerin could play a role not only in normal renal development but also in oncogenesis and regeneration.

Molecular cloning and functional expression of gicerin, a novel cell adhesion molecule that binds to neurite outgrowth factor.

Gicerin is an integral membrane glycoprotein of about 82 kd that is transiently expressed in the developing CNS. Gicerin was first identified as a binding protein for neurite outgrowth factor (NOF), a member of the laminin family of extracellular matrix proteins. By isolating and sequencing a gicerin cDNA, we have found that this protein is a novel member of the immunoglobulin superfamily. The deduced protein (584 amino acids) consists of five immunoglobulin-like loop structures in an extracellular domain, a single transmembrane region, and a short cytoplasmic tail. Cells transfected stably with gicerin cDNA adhered to NOF and aggregated with each other, indicating that gicerin exhibits both heterophilic and homophilic adhesion activities.

The alpha-helical rod domain of human lamins A and C contains a chromatin binding site.

We examined regions of human lamins A and C involved in binding to surfaces of mitotic chromosomes. An Escherichia coli expression system was used to produce full-length lamin A and lamin C, and truncated lamins retaining the central alpha-helical rod domain (residues 34-388) but lacking various amounts of the amino-terminal 'head' and carboxy-terminal 'tail' domains. We found that lamin A, lamin C and lamin fragments lacking the head domain and tail sequences distal to residue 431 efficiently assembled into paracrystals and strongly associated with mitotic chromosomes. Furthermore, the lamin rod domain also associated with chromosomes, although efficient chromosome coating required the pH 5-6 conditions needed to assemble the rod into higher order structures. Biochemical assays showed that chromosomes substantially reduced the critical concentration for assembly of lamin polypeptides into pelletable structures. Association of the lamin rod with chromosomes was abolished by pretrypsinization of chromosomes, and was not seen for vimentin (which possesses a similar rod domain). These data demonstrate that the alpha-helical rod of lamins A and C contains a specific chromosome binding site. Hence, the central rod domain of intermediate filament proteins can be involved in interactions with other cellular structures as well as in filament assembly.

Liver resection for hepatocellular carcinoma. Results of 229 consecutive patients during 11 years.

OBJECTIVE: This study analyzed the results in 229 patients with primary hepatocellular carcinoma (HCC) who were treated by radical hepatic resection in the past 11 years. SUMMARY BACKGROUND DATA: Due to marked advances in diagnostic and therapeutic methods, the therapeutic strategy for HCC has changed significantly. However, there are still many problems to be solved when hepatic resection is to be performed for HCC associated with chronic liver disease. A satisfactory result may be possible only when all of accurate operative indication, skillful surgical technique, and sophisticated postoperative management are met. METHODS: There were 188 men and 41 women. Age ranged from 32 to 79 years averaging 60.8. Underlying cirrhosis of the liver was found in 177 patients, and chronic hepatitis was found in 47 instances. Before surgery, 114 patients had 157 associated conditions; diabetes mellitus in 66, esophageal varices in 42, cholelithiasis in 22, peptic ulcer in 12, and miscellaneous in 15 cases. In addition to various types of hepatic resection, 69 patients underwent concomitant operations such as cholecystectomy, the Warren shunt, splenectomy, partial gastrectomy, and so forth. RESULTS: The 30-day (operative) mortality rate was 7.0%, and there were eight additional late deaths (3.5%). Child's class, bromosulphalein (BSP) test, and the estimated blood loss during surgery were good predictors for operative death. The cumulative 5- and 10-year survival rates for all patients were 26.4% and 19.4%, respectively. At present, 110 patients are alive; 2 more than 10 years and 21 more than 5 years. Younger age, absence of cirrhosis, smaller tumor, and postoperative chemotherapy were associated with increased survival. CONCLUSIONS: The results of hepatic resection in 229 patients with HCC were analyzed. Child's class, BSP test, and blood loss during surgery were good predictors for operative death. The 5- and 10-year survival rates were 26.4% and 19.4%, respectively. Age, liver cirrhosis, tumor size, and postoperative chemotherapy were prognostic factors. Multidisciplinary approach with liver resection, postoperative chemotherapy, and liver transplantation will be a realistic direction for the surgical treatment of HCC in future.

Liver resection in the aged (seventy years or older) with hepatocellular carcinoma.

BACKGROUND: Thirty-two patients who were 70 years of age or older underwent hepatic resection in the treatment of hepatocellular carcinoma. There were 25 men and 7 women. Age ranged from 70 to 84 years, averaging 74 +/- 3 years (mean +/- SD). Underlying liver diseases were associated in all patients but one, cirrhosis of the liver in 22, and chronic hepatitis in nine. METHODS AND RESULTS: The operative mortality rate within 1 month was 12.5%, and the overall in-hospital death rate was 18.8%. The 5-year survival rate was 17.6% for all patients and 24.3% when six hospital deaths were excluded. The Child's grade was a good predictor for early and late morbidity and death, the 5-year survival rate of patients with Child's class A disease being 30%. Retrospective comparisons were conducted between the current patients and patients younger than 50 years old who had been operated on during the same period: resectability rate, 61.5% versus 57.8%; hospital mortality rate, 18.8% versus 11.6%; and 5-year survival rate, 24.3% versus 48.6%, respectively. CONCLUSIONS: These results seem to indicate that the treatment policy of hepatocellular carcinoma in the aged should be identical to that in young people.