Distinctive role of vasohibin-1A and its splicing variant vasohibin-1B in tumor angiogenesis.

Vasohibin-1 (VASH1) was isolated as a negative-feedback regulator of angiogenesis expressed in endothelial cells (ECs). There are two transcripts of VASH1, that is, the full-length VASH1A consisting of seven exons and the splicing variant VASH1B consisting of four exons. Here, we compared the effects of VASH1A and VASH1B on tumor angiogenesis. When ECs were transfected with VASH1A or VASH1B cDNAs, VASH1B transfectants, but not VASH1A ones, induced autophagic cell death of ECs. With sonoporation, the VASH1A or VASH1B gene were transfected specifically in ECs of tumor vessels in mice. Both VASH1A and VASH1B decreased tumor vessel density and inhibited tumor growth. VASH1A normalized the remaining tumor vessels, increased their rate of perfusion, decreased tumor hypoxia and enhanced the efficacy of anticancer chemotherapy, whereas VASH1B pruned tumor vessels without causing normalization, increased tumor hypoxia and tumor necrosis and did not enhance the efficacy of anticancer chemotherapy. The alternate transfection of mice with the VASH1A and VASH1B gene showed the highest effects on antitumor activity and normalization of tumor vessels. Our present findings on VASH1A and VASH1B should provide an innovative approach that would improve the efficacy of antiangiogenic cancer therapy by balancing vascular normalization and pruning.

Size-dependent cytotoxic effects of amorphous silica nanoparticles on Langerhans cells.

Amorphous silica nanoparticles (nSPs), are widely used in medicines, cosmetics and food. However, due to their reduced particle size they are suspected to pose new risks induced by changes in biological reactivity and kinetics, which differ from those of bulk materials. In a previous study, we showed that silica particles with a diameter of 70 nm penetrated the stratum corneum (SC) of mouse skin and were taken up by living cells such as keratinocytes and Langerhans cells. To clarify the relationship between particle size, distribution and cellular response, we have evaluated size-dependent intracellular localization and cytotoxicity of silica particles, using the mouse epidermal Langerhans cell line XS52. On treatment with silica particles of diameters 70, 300, and 1000 nm, cellular uptake and cytotoxicity increased with reduction in particle size. These results suggest that smaller sized silica particles induced greater cytotoxicity against Langerhans cells, which was correlated with the quantity of particle uptake into the cells.

Cytokine-mediated induction of anti-apoptotic genes that are linked to nuclear factor kappa-B (NF-kappaB) signalling in human islets and in a mouse beta cell line.

AIMS/HYPOTHESIS: The destruction of pancreatic beta cells leading to type 1 diabetes in humans is thought to occur mainly through apoptosis and necrosis induced by activated macrophages and T cells, and in which secreted cytokines play a significant role. The transcription factor nuclear factor kappa-B (NF-kappaB) plays an important role in mediating the apoptotic action of cytokines in beta cells. We therefore sought to determine the changes in expression of genes modulated by NF-kappaB in human islets exposed to a combination of IL1beta, TNF-alpha and IFN-gamma. METHODS: Microarray and gene set enrichment analysis were performed to investigate the global response of gene expression and pathways modulated in cultured human islets exposed to cytokines. Validation of a panel of NF-kappaB-regulated genes was performed by quantitative RT-PCR. The mechanism of induction of BIRC3 by cytokines was examined by transient transfection of BIRC3 promoter constructs linked to a luciferase gene in MIN6 cells, a mouse beta cell line. RESULTS: Enrichment of several metabolic and signalling pathways was observed in cytokine-treated human islets. In addition to the upregulation of known pro-apoptotic genes, a number of anti-apoptotic genes including BIRC3, BCL2A1, TNFAIP3, CFLAR and TRAF1 were induced by cytokines through NF-kappaB. Significant synergy between the cytokines was observed in NF-kappaB-mediated induction of the promoter of BIRC3 in MIN6 cells. CONCLUSIONS/INTERPRETATION: These findings suggest that, via NF-kappaB activation, cytokines induce a concurrent anti-apoptotic pathway that may be critical for preserving islet integrity and viability during the progression of insulitis in type 1 diabetes.

Significance of HIF-1-active cells in angiogenesis and radioresistance.

Human solid tumors contain hypoxic regions that have considerably lower oxygen tension than the normal tissues. Hypoxia offers resistance to radiotherapy and anticancer chemotherapy, as well as predispose to increased tumor metastases. Furthermore, hypoxia induces hypoxia-inducible factor-1 (HIF-1), which in turn increases tumor angiogenesis. Thus, eradication of HIF-1-active/hypoxic tumor cells is very important for cancer therapy. We have previously reported that procaspase-3 fused with a von Hippel-Lindau (VHL)-mediated protein destruction motif of alpha subunit of HIF-1 (HIF-1alpha) containing Pro564, named TAT-ODD-procaspase-3 (TOP3), specifically induced cell death to hypoxic cells in vivo as well as in vitro. We now report that TOP3 also eradicates the radiation-induced HIF-1-active tumor cells. HIF-1 activity in the xenografts of human tumor cells, which express luciferase under the transcriptional control of HIF-1, were monitored and quantified daily with an in vivo bioluminescence photon-counting device. HIF-1 activity in tumors was more rapidly increased by ionizing radiation (IR) compared to untreated tumors. TOP3 efficiently decreased the HIF-1-activity in irradiated tumors as well as unirradiated ones, indicating TOP3 eradicated tumor cells with HIF-1-activity induced by IR as well as hypoxia. Eradication of HIF-1-active/hypoxic cells in the xenografts during irradiation exhibited significant suppression in angiogenesis and strong enhancement in a long-term growth suppression of tumor xenografts. These results further strengthen the argument that HIF-1-active/hypoxic cells play crucial roles in angiogenesis and radioresistance.

Multiple early bronchioloalveolar carcinomas in both lungs.

We treated a 49-year-old woman with synchronous multiple bronchioloalveolar carcinomas (BAC). Multiple bilateral 'ground-glass' opacities were detected by computed tomography (CT). We performed partial lung resection, and the pathologic diagnosis was atypical adenomatous hyperplasia. Two and one-half years later, a lesion showed enlargement, and was resected along with some others. The histopathologic diagnosis was multiple early BAC. High-resolution CT is likely to detect increasing numbers of similar cases.

A comparison of DNA copy number changes detected by comparative genomic hybridization in malignancies of the liver, biliary tract and pancreas.

Tumors arising from the liver, biliary tract and pancreas, which originate in the foregut and are in close anatomical proximity to each other, sometimes show similar histological features. No studies have focused on genetic similarities and differences between tumors of these organs. To elucidate the similarities and differences in DNA copy number alterations between tumors of these organs, we applied comparative genomic hybridization (CGH) to cancers of the liver (31 cases), biliary tract (42 cases) and pancreas (27 cases). Some alterations were common to tumors of all three organs, and some were preferential in certain types of tumor. Gains of 1q and 8q and losses of 8p and 17p were common to all tumors. In contrast, 13q14 and 16q losses were detected exclusively in hepatocellular carcinomas (HCCs; p < 0.01). The incidence of 17q21 gain and 5q loss was higher in biliary tract cancers than in the other two types (p < 0.05). Pancreatic cancers exhibited higher incidence of 5q14-q23 gain and 19p loss than tumors of other organs (p < 0.01). Gains of 7p, 7q, 12p and 20q and losses of 3p, 6q, 9p and 18q were frequent in both biliary tract and pancreatic cancers but rare in HCCs (p < 0.05). The present results suggest that although genes located at 1q, 8p, 8q and 17p are frequently involved in HCC, biliary tract and pancreatic cancer, at least some of the genes implicated in carcinogenesis are different between these three types. It is also suggested that CGH analysis is useful as a potential adjunct for the diagnosis and management of these tumors of organs that are anatomically close to one another.

[Surgical analysis for N 2 factor in non-small cell lung cancer].

In this study we analyzed 33 cases which underwent complete surgical resection to assess the role of surgery in the treatment of patients with N 2 NSCLC. The 3 year survival rate was 33.3% and the median survival time was 26.1 months. The survival curve for patients with T 3 factor was statistically worse than those with T 1 or T 2 factor. Further, the survival curve for patients with p 2 or p 3 factor was significantly lower than than that for those classified as p 0. Patients classified with p 2 or p 3 had a survival rate under 2 years. Only one patient diagnosed as T 3 achieved 3 year survival. This patient had a pm1 tumor with p 0 factor. We thereby recommend that surgery should only be performed for those N 2 NSCLC patients diagnosed as T 1 or T 2 with a classification of p1 or less.

c-IAP2 is induced by ionizing radiation through NF-kappaB binding sites.

Transcriptional promoters responsive to low doses of X-irradiation may be useful in developing a new strategy in gene therapy combined with conventional radiotherapy. The retrovirus-mediated gene trap screening identified c-IAP2 as one of genes possessing such promoters. The analysis of the cis-elements responsive to X-irradiation in c-IAP2 promoter revealed that the NF-kappaB binding sites were necessary and sufficient for the X-ray-responsiveness. We constructed the plasmid p4NFB-BAX, which had four tandem repeats of the NF-kappaB binding sites of c-IAP2 promoter (4NFB) and a suicide gene BAX under the control of 4NFB. The human tumor cells transfected with p4NFB-BAX significantly reduced the number of cells that survived 2 Gy irradiation.

Etiology and pathogenesis of aponeurotic blepharoptosis.

How and why aponeurotic blepharoptosis develops was investigated in terms of the relationship between the levator aponeurosis and Mueller's muscle functioning as the muscle spindle of the levator muscle. A total of 200 consecutive patients with moderate to severe acquired blepharoptosis completed questionnaires regarding their history of physical irritations to the eyelids, and intraoperative conditions of the levator aponeurosis and Mueller's muscle were evaluated. Several kinds of physical irritations to the eyelids were reported, such as habitual rubbing of the eyelids, contact lens usage, cataract surgery, and continuous rubbing of the eyelids while crying all night. The two main findings for aponeurosis were that it was disinserted from the tarsus, resulting in a large amount of play between the aponeurosis and the tarsus, and that the aponeurosis and Mueller's muscle were attenuated and elongated. The authors believe that rubbing may have caused disinsertion as well as attenuation and elongation of the aponeurosis, which result in transmission failures between the levator muscle and the tarsus as well as between the levator muscle and the mechanoreceptor of Mueller's muscle, leading to clinical blepharoptosis.

Comparative genomic hybridization analysis of genetic aberrations associated with development and progression of biliary tract carcinomas.

BACKGROUND: Little is known about genetic aberrations associated with development and progression of biliary tract carcinomas. METHODS: To study chromosomal aberrations associated with development and progression of biliary tract carcinomas, the authors used comparative genomic hybridization to examine 50 such carcinomas. RESULTS: Gains in part or in whole of chromosomes 1q, 8q, and 20q and losses of 5q, 8p, 9p, and 18q were detected frequently in early stage (T1/T2 classification) biliary tract carcinomas (> or = 40% of 19 early stage tumors), and they also were found in advanced stage (T3/T4 classification) tumors. In particular, loss of 9p was the most frequently observed aberration in both early stage (15 of 19; 78%) and advanced stage tumors (21 of 31; 68%). The frequencies of gains of 7p12-p14 (P < 0.003), 7p21-pter (P < 0.007), and 7q31 (P < 0.01) differed significantly between biliary tract carcinoma with and without distant metastasis. Also, gains of 5p and 19q13 and loss of 6q14-q16 were more frequent in tumors with lymph node metastasis than in those without it (P < 0.02). CONCLUSIONS: It is likely that loss of 9p is one of the genetic aberrations critical for the development of biliary tract carcinoma, whereas gains of 5p, 7p, 7q, and 19q and loss of 6q are considered later events associated with tumor progression and are thought to confer metastatic potential to biliary tract carcinomas.

Identification of a novel thioredoxin-related transmembrane protein.

We recently identified a series of transforming growth factor-beta-responsive genes in A549 human adenocarcinoma cell line by a gene trap screening method. Here we report the molecular cloning and characterization of one of these genes, designated TMX, that encodes a novel protein of 280 amino acid residues. The TMX protein possesses an N-terminal signal peptide followed by one thioredoxin (Trx)-like domain with a unique active site sequence, Cys-Pro-Ala-Cys, and a potential transmembrane domain. There are putative TMX homologs with identical active site sequences in the Caenorhabditis elegans and Drosophila genomes. Using recombinant proteins expressed in Escherichia coli, we demonstrated the activity of the Trx domain of TMX to cleave the interchain disulfide bridges in insulin in vitro. The TMX transcript is widely expressed in normal human tissues, and subcellular fractionation and immunostaining for an epitope-tagged TMX protein suggest that TMX is predominantly localized in the endoplasmic reticulum (ER). When TMX was expressed in HEK293 cells, it significantly suppressed the apoptosis induced by brefeldin A, an inhibitor of ER-Golgi transport. This activity was abolished when two Cys residues in the active site sequence were mutated to Ser, suggesting that the Trx-like activity of TMX may help relieve ER stress caused by brefeldin A.

Evaluation of the reliability of chromosomal imbalances detected by combined use of universal DNA amplification and comparative genomic hybridization.

Comparative genomic hybridization (CGH) analysis of microscopic tumor samples is allowed by universal DNA amplification using degenerate oligonucleotide primed-PCR (DOP-PCR). To evaluate the reliablity of DOP-PCR CGH, we performed DOP-PCR CGH and standard CGH in parallel using DNAs extracted from 10 malignant tumors of the hepatobiliary tract and pancreas. Similar results were obtained by both methods with a few exceptions, indicating that DOP-PCR CGH provides cytogenetic information equivalent to that obtained from standard CGH. We also investigated the sensitivity of DOP-PCR CGH using sequential dilutions of DNA from microdissected tumor cells. DOP-PCR using 100 to 800 pg of template DNA yielded successful CGH results. However, less than 50 pg of template DNA was not suitable because of the small amount of generated DNA. These findings suggest that DOP-PCR CGH is applicable for CGH analysis of tiny specimens which are too small for standard CGH. Accordingly, DOP-PCR CGH analysis may become a useful method in clinical laboratory examination.

Nonlinear density wave theory for the spiral structure of galaxies.

The theory of nonlinear waves for plasmas has been applied to the analysis of the density wave theory of galaxies which are many-body systems of gravity. A nonlinear Schrödinger equation has been derived by applying the reductive perturbation method on the fluid equations that describe the behavior of infinitesimally thin disk galaxies. Their spiral arms are characterized by a soliton and explained as a pattern of a propagating nonlinear density wave.

Calcium concentration and artificial precipitates in human pancreatic juice.

We studied the role of the increase in the calcium concentration in pure pancreatic juice of alcoholic noncalcified chronic pancreatitis. Pure pancreatic juice was obtained endoscopically. The pancreatic juice from patients with chronic pancreatitis was adjusted to pH 7.5; then the calcium concentration was adjusted to 0.4, 2.9, 5.4, or 10.4 mmol/L. Artificial precipitates were produced by incubation of the samples at 37 degrees C for 6 hours. Proteins in the artificial precipitates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein patterns were compared with the patterns of natural protein plugs from patients with chronic pancreatitis. The amount of the precipitate increased as the added calcium increased. The protein patterns of SDS-PAGE of the artificial precipitates were similar to those of protein plugs. Albumin, a-amylase, lipase, trypsinogen, and chymotrypsinogen were identified by immunoblotting both in the precipitate and in the protein plug. The increased calcium concentrations in pancreatic juice induced the formation of precipitates whose protein composition was similar to that of protein plugs. An increased calcium concentration in human pancreatic juice may play an important role in the pathogenesis of protein plugs.

Giant noninflammatory and nonatherosclerotic coronary arterial aneurysm in the left main trunk assessed by intravascular ultrasound imaging--a case report.

A case of a giant noninflammatory and nonatherosclerotic coronary arterial aneurysm in the left main trunk of a 69-year-old female is reported. Preoperative intravascular ultrasound (IVUS) images were helpful for visualizing the morphologic and histologic features of the coronary aneurysm. They were also useful for determining the etiologic background and surgical procedure.

Transient over-expression of NGFI-A gene suppresses NGF-induced neurite outgrowth in PCI2 cells.

Using an inducible gene expression system (Tet-ON system), the role of NGFI-A gene during the neuronal differentiation of PCI2 cells was examined. When NGFI-A was transiently over-expressed, no obvious effects on cell proliferation or neurite outgrowth were observed. Interestingly, however, NGFI-A over-expression resulted in significant retardation in NGF-induced neurite outgrowth. Similar suppressive effects were observed also on the v-K-ras-induced neurite outgrowth. These results raise the possibility that NGFI-A protein may play some negative role in NGF signaling.

Identification of a series of transforming growth factor beta-responsive genes by retrovirus-mediated gene trap screening.

Transforming growth factor beta (TGF-beta) plays important roles in the regulation of proliferation, differentiation, apoptosis, and carcinogenesis. To identify genes responsible for maintaining the phenotype induced by TGF-beta, we performed a retrovirus-mediated gene trap screening designed to isolate TGF-beta-responsive genes in human lung carcinoma cell line A549. After screening 249 trap lines, 21 were found to express the reporter beta-galactosidase gene in a TGF-beta-responsive manner. Interestingly, in large proportions of these trap lines, the reporter gene was responsive also to phorbol ester and was suppressed by gamma interferon. Fragments of all these trapped genes were recovered by 5'- and 3'-rapid amplification of cDNA ends (RACE), and in 15 out of 21 cases (71%), the TGF-beta responsiveness of the endogenous genes was confirmed by RNA blot hybridization. In at least five cases, the TGF-beta-induced upregulation was found to be cycloheximide resistant, suggesting the roles of the genes in the TGF-beta-induced primary responses. Sequence analyses revealed that 43% (9 of 21) of the trapped genes were novel and that the remainder included genes previously reported to be upregulated by TGF-beta, such as epidermal growth factor receptor and beta1 integrin, documenting the validity of this approach. Other known genes include the ones encoding the proteins associated with cell proliferation (ribosomal proteins S15a, hNRP/NAP-1, and lipocortin II), focal adhesions (paxillin), and transcriptional regulation (thyroid hormone receptor activator molecule 1 [TRAM-1]).

Role of TGF-beta in EGF-induced transformation of NRK cells is sustaining high-level EGF-signaling.

We have been isolating and analyzing NRK cell mutants, which fail to transform by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. One such mutant, R14, can respond to the growth inhibitory signal of TGF-beta to the same extent as parental NRK but fail to respond to the growth stimulatory signal of EGF. This mutant has a defect in EGF receptor (EGFR) expression. When R14 mutant expressed a high level of EGFR, however, EGF not only induced proliferation in this mutant but also induced transformation without the aid of TGF-beta. These findings suggest that the major role of TGF-beta in this transformation system should be to counteract the ligand-dependent down-regulation of EGFR, thereby sustaining high-level EGF-signaling.

Effects of ebselen on cerebral ischemia and reperfusion evaluated by microdialysis.

Since ebselen is known to have glutathione peroxidase-like activity and inhibitory effects on lipoxygenase and cyclo-oxygenase, we investigated its protective effects against cerebral ischemia in the rat using microdialysis. Ebselen was given through a gastric tube 30 min before occlusion in the experimental groups. Ischemia was induced using 4-vessel occlusion either transiently (20-min occlusion of the arteries followed by reperfusion), or over a prolonged period (120-min occlusion). Extracellular lactate, pyruvate and purine catabolites were sampled using microdialysis and measured by high performance liquid chromatography. During ischemia, the level of lactate, adenosine, inosine and hypoxanthine in the control group increased markedly. The lactate: pyruvate ratio increased during ischemia and decreased after reperfusion. Although the level of lactate and adenosine decreased immediately after reperfusion, those of inosine and hypoxanthine showed delayed decrease. Ebselen reduced the maximum values of lactate and purine catabolites significantly and markedly in transient ischemia. Although it reduced the values significantly in prolonged ischemia, the decrements were less marked than those in transient ischemia. Based on these results we consider ebselen to protect against ischemic metabolic changes and to accelerate the recovery during reperfusion.