BACKGROUND.Tissue factor (TF), a cell surface receptor of factor VIIIVIIa, was

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the ''A'' fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.

Neurofibromin, the neurofibromatosis type 1 (NF1) gene product, contains a central domain homologous to a family of proteins known as Ras-GTPase-activating proteins (Ras-GAPs), which function as negative regulators of Ras. The loss of neurofibromin function has been thought to be implicated in the abnormal regulation of Ras in NF1-related pathogenesis. In this study, we found a novel role of neurofibromin in neuronal differentiation in conjunction with the regulation of Ras activity via its GAP-related domain (GRD) in neuronal cells. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after NGF stimulation, which were correlated with the down-regulation of Ras activity during neurite elongation. Interestingly, the NF1-GAP increase was due to the induction of alternative splicing of NF1-GRD type I triggered by the NGF-induced Ras activation. Dominantnegative (DN) forms of NF1-GRD type I significantly inhibited the neurite extension of PC12 cells via regulation of the Ras state. NF1-GRD-DN also reduced axonal and dendritic branching/extension of rat embryonic hippocampal neurons. These results demonstrate that the mutual regulation of Ras and NF1-GAP is essential for normal neuronal differentiation and that abnormal regulation in neuronal cells may be implicated in NF1-related learning and memory disturbance. Neurofibromatosis type 1 (NF1)1 is one of the most common autosomal dominantly inherited disorders, with an incidence of about 1 in 3500 individuals (1). The NF1 hallmark is the development of benign tumors of the peripheral nervous system and the increased risk of developing malignancies. The NF1 phenotype is highly variable; it affects several organ systems, including bones, skin, irises, and central nervous system manifested as gliomas and learning disabilities. The NF1 gene lies on chromosome 17q11.2 and encodes neurofibromin, a large 2818-amino acid protein (2). Since the majority of NF1 gene mutations frequently found in NF1 patients prevent intact neurofibromin expression, functional disruption of neurofibromin is potentially relevant to the expression of some or all of the multiple abnormalities that occur in NF1 patients (3).A region centered around the 360 amino acids encoded by the NF1 gene shows significant homology to the known catalytic domains of mammalian Ras GTPase-activating protein (p120GAP) and is also similar to yeast IRA1/2 proteins, which interact with Ras and mediate hydrolysis of Ras-bound GTP to GDP, resulting in Ras protein inactivation. The GAP-related domain of the NF1 gene product (NF1-GRD) also stimulates Ras GTPase and consequently inactivates Ras protein (4, 5).Two different isoforms, type I and type II, which are formed by alternative splicing, have been identified in the NF1-GRD region. Type II contains an additional 63-bp insertion (exon 23a) that encodes 21 amino acids in the center of NF1-GRD type I (6). The specific expression patterns of the two isoforms have been studied in several organs and cells (7,8) and pro...

A novel method of in vivo targeted gene transfer to intenthelial cells as early as day 1 and lasted until day 21. The tionally selected areas of the corneal endothelium was most intense gene expression was observed on days 1 and developed. Plasmid DNA with the lacZ gene coding for ␤-3 (5.21% on day 1 and 6.45% on day 3). The expression galactosidase was injected into the anterior chamber of of ␤-galactosidase on day 3 was most evident following adult Wistar rats, and eight pulses of electricity at intendelivery of 20 V electric pulses (0.09% at 5 V, 0.03% at sities ranging from 5 to 40 V/cm were delivered for 50 ms 10 V, 6.45% at 20 V). ␤-Galactosidase expression was limto the cornea with a specially designed electric probe in ited to the corneal endothelial cells in highly selected areas order to determine the effect of gene transfer on the corand no ␤-galactosidase expression was detected in any neal endothelial cells. Gene expression was visualized by other intra-or extraocular tissues. In addition, no cell damenzymatic color reaction using X-gal in enucleated eyes on age was apparent in the cornea and no inflammation was days 1, 3, 7, 14 and 21 after gene transfer. The treated detected in any other intraocular tissues. Thus, low-voltage eyes were then photographed and the X-gal-positive areas electric pulses successfully transferred the gene of interest were evaluated by an image analyzer. The ratios of the to highly selective areas of the corneal endothelium without areas (X-gal-positive area/area of entire corneal endoinducing any pathological changes. This targeted gene thelium × 100%) were then calculated to determine gene transfer method appears to have great potential for use in transfection efficiency. The expression of ␤-galactosidase gene therapy for ocular diseases. was clearly detected in the cytoplasm of the corneal endo-

The nature of an unruptured VADA is not highly aggressive. However, if the dissection site enlarges without the manifestation of new symptoms, it should be occluded. In patients with recurrent ischemic attacks antiplatelet therapy should be considered.

Background: 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.

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