Toru Nishi

Expression of tissue factor correlates with grade of malignancy in human glioma

Tissue factor (TF), a cell surface receptor of factor VII/VIIa, was initially recognized as an initiator of the extrinsic coagulation pathway. TF has recently been found to be expressed highly in certain types of malignant tumors. In addition, TF belongs to the interferon receptor family and is one of the immediate early genes, suggesting that TF may participate in the regulation of cell growth. However, the correlation between the expression of TF and cell growth is still unclear.

Identification of the cis‐acting region in the NF2 gene promoter as a potential target for mutation and methylation‐dependent silencing in schwannoma

Although mutational inactivation and allelic loss in the NF2 gene appear to be causal events in the majority of vestibular schwannomas, involvement of another potentially important mechanism, transcriptional inactivation, has not been investigated.

Intratumoral Delivery of Interleukin 12 Expression Plasmids with In Vivo Electroporation Is Effective for Colon and Renal Cancer

We report on an antitumor treatment involving electrogene therapy (EGT), a newly developed in vivo gene transfer method using electroporation. We carried out in vivo EGT in a subcutaneous model of CT26 colon carcinoma cells, using plasmid DNAs encoding interleukin 12 (IL-12) subunits. For this purpose, we developed two IL-12 expression systems: a cotransfer system using a plasmid encoding the IL-12 p40 subunit and a plasmid encoding the IL-12 p35 subunit, and a single-vector system using a plasmid expressing a p40-p35 fusion protein. Both transfer systems significantly inhibited the growth of CT26 tumor. Immunohistochemical analysis of IL-12 EGT-treated tumors revealed enhanced infiltration of CD8(+) cells into the tumor tissue, while reverse transcriptase-polymerase chain reaction confirmed the increased expression of interferon gamma within treated tumors. The same IL-12 EGT applied to the nude mouse model was not effective, suggesting the critical role of T cell infiltration in this treatment. The inhibitory effects revealed in experiments in which previously treated mice were rechallenged with a second inoculation of CT26 tumor cells suggested that IL-12 EGT may also establish partial systemic antitumor immunity. The growth of IL-12 EGT-treated Renca tumors, a renal cell carcinoma, was also significantly inhibited. These findings suggest that EGT of the IL-12 gene has the potential to be an effective anticancer gene therapy.

Immunocytochemical localization of the striatal enriched protein tyrosine phosphatase in the rat striatum: A light and electron microscopic study with a complementary DNA-generated polyclonal antibody

The present study concerns the immunocytochemical localization of the striatal enriched protein tyrosine phosphatase in the rat striatum. A novel molecular biology technique allowed us to produce a complementary DNA-generated polyclonal antibody raised against the non-catalytic domain of the striatal enriched protein tyrosine phosphatase, which selectively recognized the striatal enriched protein tyrosine phosphatase protein with 46,000 mol. wt on western blots. Immunocytochemical analysis with the specific antibody revealed strong striatal enriched protein tyrosine phosphatase immunoreactivity in the striatum. Light microscopy showed striatal striatal enriched protein tyrosine phosphatase-immunopositive neurons to be of medium size (mean diameter of 14.4 microns), and to comprise approximately 80% of the total neuronal population in the striatum. These cells had round, triangular or polygonal cell bodies with relatively little cytoplasm. Nerve fibers stained positively for striatal enriched protein tyrosine phosphatase were also present in the globus pallidus and the substantia nigra, and the nigral labeling on the ipsilateral side almost disappeared subsequent to cerebral hemitransection, suggesting these immunolabeled structures to be striatal projections. Double-immunofluorescence analysis demonstrated separate populations of striatal enriched protein tyrosine phosphatase-positive cells and neurons stained for parvalbumin. Also, ultrastructural study showed that the striatal enriched protein tyrosine phosphatase-positive neurons (n = 50) possessed no nuclear indentations or intranuclear inclusions. Thus, most striatal striatal enriched protein tyrosine phosphatase-positive neurons were thought to be of the medium-sized spinous type. At the light microscopic level, stained striatal neurons exhibited striatal enriched protein tyrosine phosphatase immunolabeling in their somata, dendrites and axonal processes, but not in their nuclei. Electron microscopic observation showed strong striatal enriched protein tyrosine phosphatase-immunoreactivity on the inner surface of the plasmalemma, on the outer surfaces of mitochondria and on microtubules, particularly of dendrites. A heavy deposit of immunoreaction product was also present on postsynaptic densities in labeled dendrites, while a light deposit was seen on the synaptic vesicles of nerve terminals. The characteristic distribution profile of striatal enriched protein tyrosine phosphatase suggested that the enzyme may play a role in a variety of functional properties of striatal neurons, especially in postsynaptic signaling processes and in regulation of microtubular functions. On the basis of the present findings, we propose the following conclusions: (i) a protein tyrosine phosphorylation system regulated by striatal enriched protein tyrosine phosphatase is involved in certain specialized cellular processes (e.g. signal transduction cascades) of medium-sized spinous neurons distinct from those of other neuronal subsets in the striatum; (ii) a striatal medium spiny neuron is characterized by its expression of striatal enriched protein tyrosine phosphatase and, therefore, the enzyme is useful for detection of the distinct subset of striatal cells or for tracing their axonal projection fibers in the basal ganglia.

Cloning and functional characterization of the 5'-flanking region of the human monocyte chemoattractant protein-1 receptor (CCR2) gene. Essential role of 5'-untranslated region in tissue-specific expression

The human monocyte chemoattractant protein-1 receptor designated hCCR2 is an essential co-receptor in cell entry by the human immunodeficiency virus as well as a receptor for monocyte chemoattractant protein-1, a member of the family of C-C chemokines that mediate monocyte chemotaxis. To elucidate the molecular mechanisms underlying the transcriptional regulation of hCCR2, we cloned and sequenced the hCCR2 gene; it was approximately 8 kilobase pairs in length and consisted of three exons divided by two introns. In the 5′-flanking region, there were the typical mammalian promoter consensus elements, a CAAT box and a TATA box, resulting in a single transcription initiation site. In addition, we found clustered tissue-specific cis-regulatory elements such as GATA consensus sequences, Oct-1 binding sequences, and CAAT/enhancer-binding protein binding sequences. Luciferase assays with various promoter deletions and gel mobility shift assays indicated that threecis-regulatory elements located within the region from −89 to +118 are required for basal activity in THP-1 cells. One element is an octamer sequence 36-base pair upstream from the TATA box; it binds mainly to Oct-1 and is capable of increasing transcriptional activity. The other two elements, which are tandem recognition sites of the CAAT/enhancer-binding protein family, are located in the 5′-untranslated region and account for the transcriptional activation as well as the tissue specificity of hCCR2.

Improvement of quality of life in patients surgically treated for asymptomatic unruptured intracranial aneurysms

Objective: To compare the preoperative and postoperative health-related quality of life (QOL) and psychological state of patients with asymptomatic unruptured intracranial aneurysms (ICAs) who underwent elective surgery. Methods: Out of 67 patients who underwent neck clipping of ICAs, we assessed the QOL of 61 patients using Short Form-36 (SF-36); their psychological state was rated on the Hospital Anxiety and Depression Scale (HADS) before, 3 months, and 1 and 3 years after treatment. Results: The preoperative mean scores for each of the eight SF-36 domains except bodily pain were significantly lower in the study population than in the reference population. 14 (20.9%) patients experienced surgical complications defined as neurological deterioration and/or abnormal CT findings within 30 days of the operation. Despite some complications, the QOL of all operated patients returned to the mean level of the reference population 3 years after treatment. At 3 months after surgery, the scores for psychosocial activities and general health perception were transiently below the preoperative levels. According to the HADS, the patients experienced mild anxiety before the operation; it disappeared by the third postoperative month. Conclusions: Preoperatively, patients with unruptured ICAs reported a significantly decreased QOL. It further declined transiently after elective surgery, but it returned to the mean level recorded for the reference population within 3 years. Our findings suggest that these patients derived significant QOL benefits from their surgery. Hence subjective QOL issues should be considered in deciding whether treatment-related risks and their natural history, such as their potential rupture, warrant surgery of asymptomatic unruptured ICAs.

Impairment of cell adhesion by expression of the mutant neurofibromatosis type 2 (NF2) genes which lack exons in the ERM-homology domain

Neurofibromatosis 2 (NF2) is an inherited disorder characterized by a predisposition to multiple intracranial tumors. The protein encoded by the NF2 gene has striking similarities to ezrin, radixin and moesin (ERM) proteins which link membrane proteins to the cytoskeleton. Therefore, it can be speculated that the disruption of cytoskeletal organization by alterations in the NF2 gene is involved in the development of tumors. It has been reported that the majority of NF2 mutations were nonsense or frameshift mutations that result in premature termination of translation. To facilitate the detection of these mutations, we performed protein truncation test and found that 11 of 14 NF2 patients had truncational mutations (79%). Seven of the 11 patients (64%) had a splicing abnormality which lead to absence of exons in the ERM homology domain. To examine the biological significance of the exon-missing mutations in the ERM homology domain, we expressed the wild-type (wt-NF2) and the various mutant NF2s (mu-NF2s) in a fibroblast cell line by using both liposome-mediated transfection and nuclear microinjection of the expression plasmids. The wt-NF2 showed intense punctate staining in the perinuclear cytoplasm in addition to overall staining of the submembranous area, whereas the mu-NF2s lacking exons in the ERM homology domain showed granular staining at the perinuclear region without any accumulation at the submembrane region. Microinjection of wt-NF2 cDNA into the nucleus of VA13 cells revealed that wt-NF2 protein induced a progressive elongation of cell processes. Furthermore, cells that expressed mu-NF2 had decreased adhesion, which resulted in detachment from the substratum. These findings suggested that the exon-missing mutations in the ERM-homology domain may affect cell membrane–cytoskeleton signaling and consequently disrupt cell–to–cell or cell–to–matrix interaction.

Expression of Hqk Encoding a KH RNA Binding Protein Is Altered in Human Glioma

The quaking gene family encodes single KH domain RNA‐binding proteins that play vital roles in cell differentiation, proliferation, and apoptotic processes. The human quaking gene, Hqk, maps to 6q25–q26, where cytogenetic alterations associated with a variety of human malignancies, including gliomas have been reported. To assess possible relationships of Hqk with human diseases such as glial tumors, we first isolated the Hqk gene, characterized its structure and expression pattern, and carried out mutational analysis of Hqk in primary tumor samples. The Hqk gene contains 8 exons spanning a ∼200 kb genomic region, and generating at least four alternatively spliced transcripts, Hqk–5, Hqk–6, Hqk–7 and Hqk–7B, of which Hqk–7 is abundantly expressed in brain. Analysis of primary tumors demonstrated a high incidence of expression alterations of Hqk in gliomas (30%; 6/20), but not in other tumors such as schwannomas (0/3), or meningiomas (0/8). Among the tumor samples showing expression alterations, two were devoid of all three major transcripts, one was missing only the Hqk–5 message, and only the Hqk–7 message was absent in two cases. Our results thus imply the involvement of Hqk in human glial tumor progression.

Full-length dystrophin cDNA transfer into skeletal muscle of adult mdx mice by electroporation

We showed that a LacZ expression plasmid (pCAG‐lacZ) injection followed by electroporation increased the expression of the LacZ gene in the skeletal muscles of adult mdx mice up to ninefold higher as compared with simple intramuscular DNA injection. When full‐length mouse dystrophin plasmid (pCAG‐dys) and pCAG‐lacZ were co‐transfected by electroporation, 56% of dystrophin‐positive fibers were stained for β‐galactosidase activity suggesting most of these myofibers are not revertants but transfected ones. Our data indicate that electroporation in vivo could introduce large full‐length dystrophin cDNA into skeletal muscle of adult mdx mice. Muscle Nerve 27: 237–241, 2003

Suprasellar hemangioblastoma in a patient with von Hippel-Lindau disease confirmed by germline mutation study: Case report and review of the literature

BACKGROUND Hemangioblastoma (HBL) in the suprasellar region is extremely rare. CASE DESCRIPTION A suprasellar mass was found in a 33-year-old woman with retinal HBL and bilateral adrenal pheochromocytomas. The diagnosis of von Hippel-Lindau (VHL) disease was confirmed preoperatively not only by these clinical manifestations but also by germline mutation study. The existence of VHL disease indicated a diagnosis of HBL for the suprasellar mass. The results of our mutation study indicated that this patient had type II VHL disease, suggesting that careful follow-up is essential for the early detection of renal cell carcinoma, which is often associated with type II VHL disease. Here, we summarize the previously reported features of sellar and suprasellar HBLs. CONCLUSIONS HBLs in this region may be one manifestation of VHL disease. Genetic testing of the VHL gene of our patient could provide useful information to determine appropriate medical care and management.