Interleukin-1beta induces the expression of lipocortin 1 mRNA in cultured rat cortical astrocytes.

Lipocortin 1 (LC1) has been shown to increase in neuronal damage and act as a neuroprotectant and a neurotrophic factor. IL-1beta acts as a mediator of inflammation and has been reported as a potent inducer of various neurotrophic factors including nerve growth factor and fibroblast growth factor. In this study, we investigated the relationship between LC1 and IL-1beta in cultured rat astrocytes. Time-and dose-dependent experiments of IL-1beta on rat cortical astrocytes in culture revealed that the expression of LC1 mRNA was significantly augmented by IL-1beta at 8 h, 10 ng/ml. In addition, IL-1beta evoked an extracellular secretion of LC1 without its cytotoxic effects. The effect of IL-1beta was completely abolished when we treated cells with inhibitor of mitogen-activated protein kinases (MAPKs) (PD98059) (25 microM), phospholipase A(2) inhibitor mepacrine (30 microM) and protein synthesis inhibitor cycloheximide (CHX) (10 microg/ml). This suggests that induction of LC1 by IL-1beta is through a MAPKs and phospholipaseA(2) pathway and requires protein synthesis. These results indicate that IL-1beta released in the central nervous system (CNS) injury can stimulate the transcription of the LC1 gene. Subsequent synthesis and release of LC1 may provide trophic support to neurons and modulate the action of IL-1beta in brain damage.

Mechanism of hyperploid cell formation induced by microtubule inhibiting drug in glioma cell lines.

Checkpoint mechanism plays a crucial role in ensuring genomic integrity during cell cycle. Loss of checkpoint function is known to induce genomic instability and to alter ploidy of dividing cells. In this study, we examined mechanisms of hyperploid formation in glioma cells by treatment with nocodazole, which activates spindle assembly checkpoint by inhibiting microtubule polymerization. By prolonged nocodazole treatment, U251MG human glioma cell, which has a p53 mutation, underwent transient arrest at mitosis, and subsequently exited from mitotic arrest (termed 'mitotic slippage') followed by DNA replication without cytokinesis, resulting in hyperploid formation. Additionally, the heterogeneity in the number of centrosomes per cell increased during the hyperploid formation, suggesting that these hyperploid cells have genomic instability. By employing LN382 glioma cell that has a temperature-sensitive p53 mutation, we found that the activation of p53 prevents hyperploid formation after the prolonged nocodazole treatment. Furthermore, staurosporine, an inhibitor for a broad range of serine/threonine kinases including cdc2, was found to enhance hyperploid formation in U251MG cells by accelerating the induction of mitotic slippage. Interestingly, inhibitors specific for cdc2 kinase prevented the G2 to M transition but did not accelerate mitotic slippage, suggesting that staurosporine-sensitive kinases other than cdc2 are required for maintenance of spindle assembly checkpoint. Moreover, the enhancement of hyperploid formation by staurosporine was also blocked by p53-dependent G1 checkpoint. These results suggest that abrogation of G1 checkpoint is a critical factor for formation of hyperploid cells after the mitotic slippage.

Cloning of a rat glia maturation factor-gamma (rGMFG) cDNA and expression of its mRNA and protein in rat organs.

We isolated a rat glia maturation factor-gamma(rGMFG) cDNA and examined the tissue distribution of GMFG in rat by Northern and Western blot analyses. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of the putative product is highly homologous (78.9%) to rat glia maturation factor-beta (rGMFB). Northern blot analysis indicated that a 0.9-kb mRNA is predominantly expressed in rat thymus, testis, and spleen. GMFG showed a different tissue distribution from GMFB, being present predominantly in proliferative and differentiative organs.

Retinoid-induced limb defects 1: inhibition of cell proliferation in distal mesenchyme of limb buds in rats.

The present study was undertaken to investigate the effects of all-trans-retinoic acid (RA) on cell death and limb bud growth in forelimb buds and also to examine whether these events are involved in limb bone defects induced by RA in rats. RA was given at doses of 50 and 100 mg/kg to pregnant rats on Day 12 of pregnancy. Although RA did not show teratogenecity in the 50 mg/kg group, micromelia was observed in the 100 mg/kg group in all live fetuses on Day 21 of gestation. Micromelia was characterized by high incidences of proximodistal reduction of forearm bones without reduction of the humerus. The incidence of cell death in prechondrogenic areas, which differentiate into humerus and forearm bone, significantly increased 24 h after RA treatment in not only the 100 mg/kg, but also the 50 mg/kg, group. There was no difference in the incidence of cell death in the prechondrogenic area between the two groups. These observations indicate that the bone-specific defects were not the result of cell death alone in the prechondrogenic area. We examined the effects of RA on early forelimb bud growth, which is indispensable for the morphogenesis of the forelimb. Proximodistal length and protein content were decreased significantly in the forelimb bud 24 h after RA treatment at a dose of 100 mg/kg, but not 50 mg/kg. The immunohistochemical detection of bromodeoxyuridine (BrdU) incorporated into cells showed that at a dose of 100 mg/kg, cell proliferation was reduced in the distal mesenchyme, but not in the forearm-bone prechondrocytes of the forelimb bud. As the distal margin provides the cells differentiating into the prechondrocytes of future bones in the limb bud, these observations suggested that RA-induced inhibition of cell proliferation in the distal margin resulted in a decrease of forearm-bone prechondrocytes localized at more distal sites. We conclude that RA may inhibit the chondrogenesis of forearm bones by reducing cell proliferation in the distal margin of the forelimb bud, not by increasing cell death, and that this results in reduction defects in forearm bones.

Retinoid-induced limb defects 2: involvement of TGF-beta 2 in retinoid-induced inhibition of limb bud development.

We previously demonstrated that retinoid-induced inhibition of chondrogenesis in the forelimb bud may be mediated by TGF-beta2 (1). The present study was conducted to examine whether TGF-beta2 is involved in the inhibition of forelimb bud development caused by all-trans-retinoic acid (RA). Expression of TGF-beta2 was examined immunohistochemically in forelimb buds of embryos 24 h after dosing to the mother on Day 12 of gestation in the rat. In the control and 50 mg/kg group, TGF-beta2 was expressed in the epithelium and prechondrogenic area around dead cells in the forelimb bud. In the 100 mg/kg group, a dose at which RA caused reduction defects of forearm bones, TGF-beta2 expression was observed in the distal margin of forelimb buds, in which no expression was observed in the control and 50 mg/kg group. Immunohistologic studies also indicated that in the 100 mg/kg group, the expression of TGF-beta2 was enhanced in forearm-bone prechondrocytes around the dead cells. In a whole embryo culture system, exposure to RA for 24 h reduced the proximodistal length and protein content in forelimb buds at concentrations of 3 microg/mL or more. The whole embryo culture system also showed that the expression of TGF-beta2 was induced at the concentration of 3 microg/mL in the same region as found in forelimb buds of embryos from dams administered a teratogenic dosage of RA in vivo. Local application of TGF-beta2 to the distal margin of the forelimb bud in Day 12 embryos reduced proximodistal growth and protein content in forelimb buds for 24 h in culture even without RA treatment. We also found that exogenous TGF-beta2 inhibited DNA synthesis of forelimb bud cells in culture in a concentration-dependent manner. Neutralization of TGF-beta2 with its antibody in the distal margin of forelimb buds partially prevented the RA-induced inhibition of forelimb bud growth in the whole embryo culture system. These results suggest that RA-induced TGF-beta2 in the distal margin of forelimb buds may be involved in RA-induced inhibition of forelimb bud growth via reduction of cell proliferation in the distal margin, and RA-induced TGF-beta2 in the prechondrogenic area may inhibit chondrogenesis in the future forearm bones, followed by reduction defects of the forearm bones.

CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration.

CD44 is a cell surface receptor for hyaluronate, a component of the extracellular matrix (ECM). Although CD44 has been implicated in tumor invasion and metastasis, the molecular mechanisms remain to be elucidated. Here we find that CD44 expressed in cancer cells is cleaved at the membrane-proximal region of the ectodomain and the membrane-bound cleavage product can be detected using an antibody against the cytoplasmic domain of CD44. Furthermore, we report that CD44 cleavage is mediated by a membrane-associated metalloprotease expressed in cancer cells. A tissue inhibitor of metalloproteases-1 (TIMP-1), as well as metalloprotease inhibitors, inhibit CD44 cleavage in the cell-free assay. Contrary, serine protease inhibitors enhance CD44 cleavage, and the enhancement can be prevented by pretreatment with a metalloprotease inhibitor. Thus, CD44 cleavage is regulated by an intricate balance between some proteases and their inhibitors. Interestingly, treatment with the metalloprotease blocker 1,10-phenanthroline, which strongly prevent the CD44 cleavage, suppressed RERF-LC-OK lung cancer cell migration on a hyaluronate substrate, but not on several other substrates. These results suggest that CD44 cleavage plays a critical role in an efficient cell-detachment from a hyaluronate substrate during the cell migration and consequently promotes CD44-mediated cancer cell migration. Our present data indicate that CD44, not only ECM per se, is one of the targets of pericellular proteolysis involved in tumor invasion and metastasis.

A novel human nucleoside diphosphate (NDP) kinase, Nm23-H6, localizes in mitochondria and affects cytokinesis.

Nucleoside diphosphate kinases (NDP kinases) are enzymes known to be conserved throughout evolution and have been shown to be involved in various biological events, in addition to the "housekeeping" phosphotransferase activity. We present the molecular cloning of a novel human NDP kinase gene, termed Nm23-H6. Nm23-H6 gene has been mapped at chromosome 3p21.3 and is highly expressed in heart, placenta, skeletal muscle, and some of the cancer cell lines. Recombinant Nm23-H6 protein has been identified to exhibit functional NDP kinase activity. Immunolocalization studies showed that both endogenous and inducibly expressed Nm23-H6 proteins were present as short, filament-like, perinuclear radical arrays and that they colocalized with mitochondria. Cell fractionation study also demonstrated the presence of Nm23-H6 protein in a mitochondria-rich fraction. Moreover, induction of overexpression of Nm23-H6 in SAOS2 cells, using the Cre-loxP gene activation system, resulted in growth suppression and generation of multinucleated cells. Flow cytometric analysis also demonstrated that the proportion of cells with more than 4N DNA content increased to 28.1% after induction of Nm23-H6, coinciding with the appearance of multinucleated cells. These observations suggest that Nm23-H6, a new member of the NDP kinase family, resides in mitochondria and plays a role in regulation of cell growth and cell cycle progression.

Identification of a novel human homolog of the Drosophila dlg, P-dlg, specifically expressed in the gland tissues and interacting with p55.

We have identified a novel human homolog of the Drosophila dlg tumor suppressor gene, termed P-dlg, which has been mapped at chromosome 10q23. Unlike other human dlg homologs, P-dlg is expressed in placenta and various gland tissues but not in brain. The P-dlg protein is localized at the plasma membrane and cytoplasm, and it is expressed in the gland epithelial cells in normal prostate tissue but not in prostate cancer cell lines. Furthermore, we identified interaction between P-dlg and p55 palmitoylated membrane protein by yeast two-hybrid screening. These findings suggest that P-dlg forms a complex with p55 at the plasma membrane and plays roles in maintaining the structure of epithelial cells and transmitting extracellular signals to the membrane and cytoskeleton, which may negatively regulate cell proliferation.

Identification of a human homolog of the Drosophila neuralized gene within the 10q25.1 malignant astrocytoma deletion region.

The loss of chromosome 10 is the most frequent genetic alteration found in malignant astrocytomas. In particular, the long arm of chromosome 10 was previously reported to have two or more common deletion regions where tumor suppressor genes may be located. In this study, we performed deletion mapping of 44 malignant astrocytomas using 12 microsatellite markers on chromosome 10q and demonstrated that the minimal common region of loss of heterozygosity (LOH) was present between D10S192 and D10S566 localized at 10q25.1. Subsequently, we have identified a novel gene, termed h-neu, within the region frequently deleted and found that h-neu encodes a protein with strong homology to the Drosophila neuralized (D-neu) protein. Northern blot and RT-PCR analyses revealed that h-neu mRNA was expressed at very low levels in human malignant astrocytoma tissues and the majority of glioma cell lines examined, while normal brains expressed h-neu transcript. Furthermore, DNA sequencing analysis of the h-neu transcript revealed one of the glioma cell lines, U251MG, had a single nucleotide substitution which resulted in an amino acid change from glycine (GGC) to serine (AGC) at codon 253. The D-neu gene is known to serve a critical function in neurogenesis in Drosophila, and loss-of-function mutations produce hyperplasia of primitive neuronal cells. These observations led us to hypothesize that h-neu gene plays a role in determination of cell fate in the human central nervous system and may act as a tumor suppressor whose inactivation could be associated with malignant progression of astrocytic tumors.

Intracranial intraparenchymal schwannoma: report of three cases.

Intracranial intraparenchymal schwannomas are rare. We report three patients with an intracranial intraparenchymal schwannoma and discuss the clinical and neuroradiological aspects of this particular tumour. The patients were a 21-year-old male, a 64-year-old female and a 17-year-old male. The tumours were located in cerebrum in two patients and the cerebellum in one patient. Computerized tomography (CT) scans demonstrated a slightly high density area with homogeneous enhancement by contrast medium. Magnetic resonance imaging (MRI) showed slightly low signal intensity on the T1-weighted image, high or mixed signal intensity on the T2-weighted image and homogeneous enhancement by gadolinium diethylene triamine penta-acetic acid (Gd-DTPA). Radiological studies revealed cystic components in 2 of the 3 patients. All tumours were firm, well-demarcated, and completely removed. The diagnosis of schwannoma was derived from histological and immunohistochemical studies in all 3 cases; 2 cases were also examined by electron microscopy.

Relation of TGF-beta 2 to inhibition of limb bud chondrogenesis by retinoid in rats.

A newly synthesized retinoid, Am-80 (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl] carbamoxyl) benzoic acid, induced limb reduction defects in rats in vivo. The present study was designed to investigate the mechanisms of its teratogenic action and also to examine whether transforming growth factor-beta (TGF-beta) is involved in limb reduction defects by using a rat limb bud cell culture system. Am-80 inhibited DNA synthesis and chondrogenesis of limb bud cells from day 12 embryos at concentrations of 1-4 ng/ml in a concentration-dependent manner. Quantities of TGF-beta 1 and 2 in culture medium from limb bud cells treated with Am-80 were evaluated using sandwich ELISAs. Although TGF-beta 1 was not detected in conditioned medium with or without Am-80, TGF-beta 2 increased in a concentration-dependent manner at drug concentrations that inhibited DNA synthesis and chondrogenesis. In addition, when TGF-beta 2 was exogenously added to limb bud cell culture medium, the synthesis of glycosaminoglycan (GAGs) was reduced even without Am-80 treatment. Neutralization of TGF-beta 2 with antibody prevented the inhibition of GAGs synthesis induced by Am-80. These results suggest that TGF-betas, especially beta 2, are involved in the inhibition of chondrogenesis caused by Am-80 in limb bud cells.

An unusual type of Hangman's fracture with cord compression: a case report.

An unusual case of hangman's fracture with neurologic sequelae is described in a 43-year-old woman. The fracture involved the body of C-2 with gross dislocation of the anterior part of C-2 and C-1 forward on C-3, associated with severe cord compression. The patient was successfully treated with a decompression of the foramen magnum and C-1 laminectomy, accompanying in situ fusion. The images obtained by computed tomography and magnetic resonance imaging were useful in understanding the true nature of the injury and planning the appropriate surgical treatment.

[Dilated pericerebellar fluid space in patients with chronic subdural hematoma].

It is not uncommon to observe the dilation of the pericerebellar fluid space (PCFS) on CT in patients with chronic subdural hematoma (CSDH). CT scans of 92 patients with CSDH proven by surgery were reviewed with respect to the dilatation of PCFS and we evaluated the incidence of dilated PCFS and the relationship between PCFS and other factors. There were 68 males and 24 females. Patients ranged in age from 20 to 90 years (mean 65.2 years). Another 50 patients without CSDH were also reviewed as a control group. A new PCFS grading based on the CT findings was proposed, divided into 3 grades as follows. In grade 0, no PCFS could be seen on CT scans. In grade 1, PCFS could be detected along the posterior aspect of the petrous pyramid, and in grade 2, PCFS could be seen not only along the posterior aspect of the petrous pyramid but also under the tentorium cerebelli. The dilation of PCFS was seen in 78 patients (84.8%) out of the 92 cases. In 50 patients without CSDH (control group), the dilatation of PCFS was noted only in 6 (12%). The dilatation of PCFS was almost always seen on the same side as the CSDH. Among many factors, the significant factor was the degree of the midline shift, the bigger the midline shift caused by CSDH, the larger was the dilated PCFS. Although the mechanism of the dilated PCFS in patients with CSDH is not clear, it is postulated that the mechanism is caused by CSF flow disturbance, compression or adhesion of the subarachnoid space due to CSDH.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous subperiosteal hematoma of the orbit--case report.

A 62-year-old female was admitted with complaints of sudden proptosis of the left eye and severe left orbital pain without history of trauma or previous illness. Computed tomography and magnetic resonance imaging demonstrated a biconvex extraconal hematoma in the upper part of the left orbit. Surgical exploration of the orbit revealed the presence of a subperiosteal hematoma without a causative lesion. Such spontaneous subperiosteal hematoma of the orbit is extremely rare, but may result from an occult vascular disorder.