Internalization of a novel, huge lectin from Ibacus novemdentatus (slipper lobster) induces apoptosis of mammalian cancer cells.

An N-acetyl sugar-binding lectin (termed iNoL) displaying cytotoxic activity against human cancer cells was isolated from the slipper lobster Ibacus novemdentatus (family Scyllaridae). iNoL recognized monosaccharides containing N-acetyl group, and glycoproteins (e.g., BSM) containing oligosaccharides with N-acetyl sugar. iNoL was composed of five subunits (330, 260, 200, 140, and 30 kDa), which in turn consisted of 70-, 40-, and 30-kDa polypeptides held together by disulfide bonds. Electron microscopic observations and gel permeation chromatography indicated that iNoL was a huge (500-kDa) molecule and had a polygonal structure under physiological conditions. iNoL displayed cytotoxic (apoptotic) effects against human cancer cell lines MCF7 and T47D (breast), HeLa (ovarian), and Caco2 (colonic), through incorporation (internalization) into cells. The lectin was transported into lysosomes via endosomes. Its cytotoxic effect and incorporation into cells were inhibited by the co-presence of N-acetyl-D-mannosamine (ManNAc). Treatment of HeLa cells with iNoL resulted in DNA fragmentation and chromatin condensation, through activation of caspase-9 and -3. In summary, the novel crustacean lectin iNoL is incorporated into mammalian cancer cells through glycoconjugate interaction, and has cytotoxic (apoptotic) effects.

Sialyl-glycoconjugates in cholesterol-rich microdomains of P388 cells are the triggers for apoptosis induced by Rana catesbeiana oocyte ribonuclease.

SBL/RC-RNase was originally isolated from frog (Rana catesbeiana) oocytes and purified as a novel sialic acid-binding lectin (SBL) that displayed strong anti-cancer activity. SBL was later shown to be identical to a ribonuclease (RC-RNase) from oocytes of the same species. The administration of SBL/RC-RNase induced apoptosis (with nuclear condensation and DNA fragmentation) in mouse leukemia P388 cells but did not kill umbilical vein endothelial or fibroblast cells derived from normal tissues. The cytotoxic activity of SBL/RC-RNase was inhibited by desialylation of P388 cells and/or the co-presence of free bovine submaxillary mucin. FACS analysis showed that SBL/RC-RNase was incorporated into cells after attachment to cholesterol-rich microdomains. Addition of the cholesterol remover methyl-ß-cyclodextrin reduced SBL/RC-RNase-induced apoptosis. Apoptosis occurred through the caspase-3 pathway following activation of caspase-8 by SBL/RC-RNase. A heat shock cognate protein (Hsc70) and a heat shock protein (Hsp70) (each 70 kDa) on the cell membrane were shown to bind to SBL/RC-RNase by mass spectrometric and flow cytometric analyses. Quercetin, an inhibitor of Hsc70 and Hsp70, significantly reduced SBL/RC-RNase-induced apoptosis. Taken together, our findings suggest that sialyl-glycoconjugates present in cholesterol-rich microdomains form complexes with Hsc70 or Hsp70 that act as triggers for SBL/RC-RNase to induce apoptosis through a pathway involving the activation of caspase-3 and caspase-8.

Production of active MMP7 in E. coli and its application for metalloproteinase inhibitors screening.

MMP-7 is the smallest metalloproteinase. Its unregulated activities and existence in serum are recently known to be tightly related with life-threatening disease such as cardiac disease and several cancers. The protein production is thought to be useful for its characterization and antibody generation. Although many attempts at bacterial expressions have been conducted, they were recovered as insoluble and inactive protein. In this study, after soluble expression, single-step purification and conversion to active protease, it was applied for the screening secretory metalloproteinase inhibitors in conditioned media of human cancer cells.

Prognostic analysis of pulmonary adenocarcinoma subclassification with special consideration of papillary and bronchioloalveolar types.

AIMS: The third edition of the World Health Organization (WHO) classification of lung tumours has been published and is expected to become the standard nomenclature. The aim of this study was to assess the usability and prognostic significance of the WHO classification in comparison with other recent classifications. METHODS AND RESULTS: One hundred and forty-seven resected pulmonary adenocarcinoma cases were reviewed and histologically classified according to the WHO classification (1999) and the classification by Noguchi (1995). Papillary carcinomas as described by Silver and Askin (1997) were also identified. Since the papillary type in the WHO classification is not strictly defined, we compared the following two kinds of WHO classification: (i) WHO-N; WHO classification adopting Noguchi Type F as the definition of the papillary type, namely, pure papillary adenocarcinoma without a bronchioloalveolar component; (ii) WHO-SA; WHO classification adopting papillary carcinoma by Silver and Askin as the definition of the papillary type, namely, tumour with papillary structure constituting at least 75% of the lesion. The bronchioloalveolar carcinoma of the WHO classification showed a better prognosis than other subtypes in both overall and Stage I disease limited survival analysis. In analysis limited to Stage III disease, only the papillary type of WHO-SA showed a significantly worse prognosis. CONCLUSIONS: WHO-SA is recommended for prognostic correlation.

Periodate-treated, non-anticoagulant heparin-carrying polystyrene (NAC-HCPS) affects angiogenesis and inhibits subcutaneous induced tumour growth and metastasis to the lung.

Periodate-treated, non-anticoagulant heparin-carrying polystyrene consists of about ten periodate-oxidized, alkaline-degraded low molecular weight-heparin chains linked to a polystyrene core and has a markedly lower anti-coagulant activity than heparin. In this study, we evaluated the effect of non-anticoagulant heparin-carrying polystyrene on tumour growth and metastasis. Non-anticoagulant heparin-carrying polystyrene has a higher activity to inhibit vascular endothelial growth factor-165-, fibroblast growth factor-2- or hepatocyte growth factor-induced human microvascular endothelial cell growth than heparin, ten periodate-oxidized-heparin and ten periodate-oxidized-low molecular weight-heparin, which is probably due to the heparin-clustering effect of non-anticoagulant heparin-carrying polystyrene. Non-anticoagulant heparin-carrying polystyrene inhibited human microvascular endothelial cell, B16 melanoma and Lewis lung cancer cell adhesion to Matrigel-coated plates. Non-anticoagulant heparin-carrying polystyrene also showed strong inhibitory activities in the tubular formation of endothelial cells on Matrigel and B16-melanoma and Lewis lung cancer cell invasion in a Matrigel-coated chamber assay. In vivo studies showed that growth of subcutaneous induced tumours and lung metastasis of B16-melanoma and Lewis lung cancer cells were more effectively inhibited by non-anticoagulant heparin-carrying polystyrene than ten periodate-oxidized-heparin and ten periodate-oxidized-low molecular weight-heparin. Furthermore, non-anticoagulant heparin-carrying polystyrene markedly reduced the number of CD34-positive vessels in subcutaneous Lewis lung cancer tumours, indicating a strong inhibition of angiogenesis. These results suggest that non-anticoagulant heparin-carrying polystyrene has an inhibitory activity on angiogenesis and tumour invasion and may be very useful in cancer therapy.

Expression of matrix metalloproteinases in invasive pulmonary adenocarcinoma with bronchioloalveolar component and atypical adenomatous hyperplasia.

To investigate the association between the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) and the clinicopathological features in lepidic and invasive components of adenocarcinoma of the lung, we performed immunostaining for type IV collagen, various MMPs, and TIMPs in 27 cases of invasive adenocarcinomas and 5 cases of atypical adenomatous hyperplasia of alveolar epithelial cells (AAH). Mean extent of lepidic growth was 61% and the survival was significantly better in cases with 50% or more lepidic component. The preservation of type IV collagen in lepidic areas correlated inversely with lymphatic or vascular invasion (P = 0.02 and 0.002, respectively). Five-year survival was reduced in cases showing destruction of type IV collagen (P = 0.004) or expression of MMP-2 (P = 0.008) in lepidic areas. MMP-2 co-localized with MT-1-MMP (its activating enzyme) and TIMP-2 in neoplastic cells. Reactivity for other MMPs and TIMPs did not correlate with destruction of type IV collagen or prognosis. Type IV collagen was preserved in all cases of AAH. MMP-2, but not MT-1-MMP, was expressed in two of the five cases of AAH. Immunostaining for type IV collagen MMP-2 is useful in evaluating the prognosis of the lung.

Experimental evaluation of photocrosslinkable chitosan as a biologic adhesive with surgical applications.

BACKGROUND: In various surgical cases, effective tissue adhesives are required for both hemostasis (eg, intraoperative bleeding) and air sealing (eg, thoracic surgery). We have designed a chitosan molecule (Az-CH-LA) that can be photocrosslinked by ultraviolet (UV) light irradiation, thereby forming a hydrogel. The purpose of this work was to evaluate the effectiveness and safety of the photocrosslinkable chitosan hydrogel as an adhesive with surgical applications. METHODS: The sealing ability of the chitosan hydrogel, determined as a bursting pressure, was assessed with removed thoracic aorta, trachea, and lung of farm pigs and in a rabbit model. The carotid artery and lung of rabbits were punctured with a needle, and the chitosan hydrogel was applied to, respectively, stop the bleeding and the air leakage. In vivo chitosan degradability and biologic responses were histologically assessed in animal models. RESULTS: The bursting pressure of chitosan hydrogel (30 mg/mL) and fibrin glue, respectively, was 225 +/- 25 mm Hg (mean +/- SD) and 80 +/- 20 mm Hg in the thoracic aorta; 77 +/- 29 mm Hg and 48 +/- 21 mm Hg in the trachea; and in the lung, 51 +/- 11 mm Hg (chitosan hydrogel), 62 +/- 4 mm Hg (fibrin glue, rubbing method), and 12 +/- 2 mm Hg (fibrin glue, layer method). The sealing ability of the chitosan hydrogel was stronger than that of fibrin glue. All rabbits with a carotid artery (n = 8) or lung (n = 8) that was punctured with a needle and then sealed with chitosan hydrogel survived the 1-month observation period without any bleeding or air leakage from the puncture sites. Histologic examinations demonstrated that 30 days after application, a fraction of the chitosan hydrogel was phagocytosed by macrophages, had partially degraded, and had induced the formation of fibrous tissues around the hydrogel. CONCLUSIONS: A newly developed photocrosslinkable chitosan has demonstrated strong sealing ability and a great potential for use as an adhesive in surgical operations.

Computed tomography-guided frameless stereotactic radiotherapy for stage I non-small cell lung cancer: a 5-year experience.

PURPOSE: Stereotactic radiotherapy (SRT) is highly effective for brain metastases from non-small-cell lung cancers (NSCLCs). As such, primary lesions of NSCLC may also be treated effectively by similar focal high-dose SRT. METHODS AND MATERIALS: Between October 1994 and June 1999, 50 patients with pathologically proven T1-2N0 M0 NSCLC were treated by CT-guided frameless SRT. Of these, 21 patients were medically inoperable and the remainder were medically operable but refused surgery. In most patients, SRT was 50-60 Gy in 5-10 fractions for 1-2 weeks. Eighteen patients also received conventional radiotherapy of 40-60 Gy in 20-33 fractions before SRT. RESULTS: With a median follow-up period of 36 months (range 22-66), 30 patients were alive and disease free, 3 were alive with disease, 6 had died of disease, and 11 had died intercurrently. Local progression was not observed on follow-up CT scans in 47 (94%) of 50 patients. The 3-year overall survival rate was 66% in all 50 patients and 86% in the 29 medically operable patients. The 3-year cause-specific survival rate of all 50 patients was 88%. No definite adverse effects related to SRT were noted, except for 2 patients with a minor bone fracture and 6 patients with temporary pleural pain. CONCLUSIONS: SRT is a very safe and effective treatment for Stage I NSCLC. Additional studies involving a larger patient population and longer follow-up periods are warranted to assess this new treatment for early-stage lung cancer.

Estimation of anti-platelet drugs on human platelet aggregation with a novel whole blood aggregometer by a screen filtration pressure method.

1. The effects of anti-platelet drugs on human whole blood aggregation were evaluated using a novel whole blood aggregometer by a screen filtration pressure (SFP) method. 2. The SFP whole blood aggregometer was found to successfully detect whole blood aggregation induced by ADP, collagen and TRAP by measuring the SFP of blood samples. The platelet aggregation threshold index (PATI), the concentration of agonist required with an inducing pressure rate of 50%, varied time-dependently after collection of blood. High values for ADP and collagen were noted immediately after blood collection, suggesting low aggregation activity of platelets, and gradually increase thereafter. 3. Cilostazol (phosphodiesterase 3 inhibitor), dipyridamole, aspirin and tirofiban all inhibited whole blood aggregation in vitro. Inhibitory effects of cilostazol and dipyridamole, but not tirofiban, were markedly enhanced 6 or 7 fold by long pre-incubation (60 min), compared with short pre-incubation (2 min). Such enhancement was only observed with ADP- and not collagen-induced whole blood aggregation. A similar phenomenon was also observed for aggregation with platelet rich plasma (PRP). Cilostazol inhibition of ADP-induced platelet aggregation was more potent with PRP than whole blood (PATI(200)=3.80+/-0.95 microM for whole blood; 2.04+/-0.61 microM for PRP). Inhibitory effects of dipyridamole were attenuated in PRP without erythrocytes. 4. These results demonstrate that the SFP aggregometer can sensitively detect anti-platelet aggregatory effects of various kinds of drugs. So that it is a useful tool for evaluation of anti-platelet drugs.

Lung carcinoma with rhabdoid cells: a clinicopathological study and survival analysis of 14 cases.

AIMS: We determined the clinicopathological features of primary lung carcinomas with rhabdoid cells by defining the immunophenotype of rhabdoid cells and analysing survival. METHODS AND RESULTS: Rhabdoid cells are distinctive in having an eccentric nucleus and a large intracytoplasmic inclusion on routinely stained sections. Based on the number of rhabdoid cells, 45 cases of large cell carcinoma were divided into the following three types: lung tumour with a rhabdoid phenotype (LTRP) (n=4), lung carcinoma with a small number of rhabdoid cells (LCSR) (n=10), large cell carcinoma containing no rhabdoid cells (LCNR) (n=31). LTRP is composed of at least 10% rhabdoid cells. In LCSR the percentage of rhabdoid cells is less than 10%. LTRP and LCSR are associated with locally advanced disease. Immunohistochemical stains were positive for epithelial markers in all LTRP and eight LCSR, for neuroendocrine markers in one LTRP and three LCSR. The outcome is worse for patients with LTRP than LCSR or LCNR. LCSR shows a trend close to LCNR. Stage-matched survival analysis, however, revealed no statistically significant difference among the histological subtypes. CONCLUSIONS: Rhabdoid cells are heterogeneous except for epithelial markers and vimentin positivity. Less than 5% of rhabdoid cells has a negligible effect on prognosis.

Characterization of whole blood aggregation with a new type of aggregometer by a screen filtration pressure method.

A new type of platelet aggregometer of whole blood, based on the screen filtration pressure method, has been developed and characterized. It measures resistance of flow of whole blood samples through a screen of microsieve with 30-microm(2) openings and provides pressure rate as an index of platelet aggregation. On optical microscopic observation, platelet aggregates, but not fibrin fibers, were found to be trapped on microsieves, and the pressure rate and protein amounts on microsieves are correlated. The aggregometer showed good reproducibility for investigations performed on different days. The time course of pressure rates indicated a bell curve change, where the pressure rate was very low immediately after blood collection and gradually increased up to 60 min thereafter. Use of the aggregometer was able to confirm that orally administered aspirin inhibits ADP- and collagen-induced whole blood aggregation as well as platelet aggregation. The results suggest that this platelet aggregometer might be useful in research and clinical diagnosis of thrombotic diseases.

Comparative study of blood group-recognizing lectins toward ABO blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides.

Binding specificities of ABO blood group-recognizing lectins toward blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides were studied by lectin-blotting analysis, enzyme linked immunosorbent assay and lectin-conjugated agarose column chromatography. Human serum albumin conjugated with A- and B-trisaccharides was clearly recognized by Helix pomatia (HPA), Phaseolus lunatus, Dolichos biflorus agglutinins, and Griffonia simplicifolia I agglutinin B(4), respectively. Almost the same results were obtained for human group A and B ovarian cyst and A-active hog gastric mucins, but Glycine max agglutinin only reacted to the group A hog mucin. When human plasma von Willebrand factor (vWF), having Asn-linked blood group antigens, was tested, HPA was highly sensitive to blood group A antigen on the vWF. Ulex europaeus agglutinin I (UEA-I) preferentially bound to the vWF from blood group O plasma. Within the GalNAc-recognizing lectins examined, a biantennary complex-type oligosaccharide having the blood group A structure retarded on an HPA-agarose column, and the affinity was diminished after digestion with alpha-N-acetylgalactosaminidase. This product bound to UEA-I agarose column. These results indicate that HPA and UEA-I are most sensitive for detection of glycoproteins possessing small amounts of blood group A and H antigens and also useful for fractionation of complex-type oligosaccharides with blood group A and H antigens, respectively.

Prognostic significance of frequent acidophilic nuclear inclusions in adenocarcinoma of the lung with immunohistochemical and ultrastructural studies.

BACKGROUND: Adenocarcinoma of the lung occasionally has acidophilic nuclear inclusions (ANIs). Some studies have reported that the incidence of ANIs was higher in well differentiated tumor types and have suggested that adenocarcinoma patients with ANIs might have a more favorable prognosis; however, to the authors' knowledge, statistically significant prognostic findings were not reported. The objective of the current study was to assess the prognostic significance of ANI in patients with pulmonary adenocarcinoma and, moreover, to characterize ANI immunohistochemically and ultrastructurally. METHODS: Surgically resected tumor specimens from 147 patients with primary pure adenocarcinoma of the lung were examined. Only obvious ANIs surrounded by a clear halo on hematoxylin and eosin-stained slides were counted; the authors classified cases with > or = 10 ANIs per 10 high-power fields (/10 HPF) as frequent-ANI cases, cases with < 10 ANIs/10 HPF as infrequent-ANI cases, and cases without ANIs as non-ANI cases in the current study. RESULTS: Nineteen frequent-ANI cases (12.9%) and 16 infrequent-ANI cases (10.9%) were found; the remaining 112 cases (76.2%) were considered to be non-ANI cases. The majority of ANIs immunohistochemically contained surfactant apoprotein and ultrastructurally corresponded to invagination of the inner nuclear membrane, showing a tubular or amorphous configuration. Frequent-ANI patients showed significantly better prognosis than the other two groups on both overall univariate analysis and univariate analysis limited to patients with International Union Against Cancer Stage I disease (P = 0.0096 and P = 0.0095, respectively). However, on the multivariate analysis only disease stage was shown to be a significant prognostic factor and frequent-ANI showed borderline significance (P = 0.0956). CONCLUSIONS: Frequent ANIs appear to be of limited value in clarifying the prognosis of patients with lung adenocarcinoma.

Telomerase activity and expression of human telomerase RNA component and human telomerase reverse transcriptase in lung carcinomas.

The aim of this study was to evaluate the usefulness of determination of telomerase activity and expression of human telomerase RNA component (hTERC) and human telomerase reverse transcriptase (hTERT) for the diagnosis of lung carcinomas. The tissues studied consisted of 115 carcinomas and adjacent nonneoplastic lung, which were removed surgically without previous chemotherapy or radiotherapy. Telomerase activity was determined using a semiquantitative polymerase chain reaction-based telomeric repeat amplification protocol (TRAP) assay. The results obtained were classified into high and low telomerase groups. Localization of expression was examined by using in situ hybridization and immunohistochemistry. The correlation between telomerase activity in lung carcinoma and clinicopathologic features, including prognosis, was investigated. Telomerase activity in lung carcinomas was detected in 107 of 115 (93%) lung carcinomas, but not in any adjacent noncancerous tissues, and was significantly higher in small cell carcinoma than in any other histologic type. This activity also was significantly higher in poorly differentiated than in well-differentiated squamous cell carcinomas and adenocarcinomas. The overall survival rate (P =.020) was significantly lower in the high telomerase group. Messenger RNAs for hTERC and hTERT were mainly detected in the cytoplasm of cancer cells by in situ hybridization, and TERT protein was localized in the nuclei of these cells by immunohistochemical staining. Determinations of telomerase activity by in situ hybridization, immunohistochemistry, and TRAP assay are useful for evaluating the diagnosis and prognosis of lung carcinomas.

Development and assessment of enzyme immunoassay for platelet-derived microparticles.

Platelet-derived microparticles (PMPs) are released from platelets through the platelet activation by high shear stress, collagen, or calcium ionophore (A23187). PMPs are observed in patients with acute myocardial infarction, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, heparin-induced thrombocytopenia and other thrombotic disorders, but the importance of circulating PMPs in the pathogenesis of these diseases is still debated. Numbers of PMPs are usually determined by flowcytometry (FCM), but easier and reproducible PMP assay systems are needed. To develop a better ELISA for PMPs, we used antibodies against the platelet antigens anti-GPIb (NNKY5-5), anti-GPIIb/IIIa (NNKY2-11, anti-CD41), anti-GPIX (KMP-9), and anti-CD9 (NNKY1-19). PMPs were detected with all combinations of these antibodies, but the ELISA having the highest and most specific absorbance was obtained with a combination of KMP-9 (capture antibody) and NNKY5-5 (detecting antibody). PMPs in blood samples were measured by ELISA and FCM. ELISA correlated with PMPs quantitated by FCM. By shaking ELISA plates during incubation, nonspecific binding of platelets was eliminated. The level of PMPs was not increased in diabetes mellitus, thrombotic thrombocytopenic purpura, antiphospholipid syndrome, or sepsis. The concentration of PMP was elevated in hemolytic uremic syndrome. Activated PMPs were absorbed to 0.8 microm filter, but circulating PMPs were not absorbed. These results suggest that activated PMPs are likely to adhere to leukocytes or endothelial cells at the activation site and that the circulating form of PMPs are likely to be a residue of activated PMPs. To detect only the activated form of PMPs, a new ELISA needs to be developed, and it will likely use a combination of antibodies that detect platelet activation markers such as P-selectin (CD62P) or activated GPIIb/IIIa.

Structure-activity relationship of mycoloyl glycolipids derived from Rhodococcus sp. 4306.

Novel mycoloyl glycolipids with short carbon chains were isolated and purified from Rhodococcus sp. 4306, a soil origin of Actinomycetales. Their chemical structures were identified as trehalose 6,6'-dimycolate (TDM), trehalose 6-monomycolate, glucose 6-monomycolate, mannose 6-monomycolate and fructose 6-monomycolate. The length of carbon chains and number of double bonds of mycolic acids were C(34), C(36)and C(38)saturated, monoenoic and dienoic molecular species, which were much shorter than those of Mycobacterium tuberculosis (C(78-88)monoenoic and dienoic). Among them, only TDM could induce prominent granulomatous inflammation of the lung and spleen in mice. By contrast, other mycoloyl glycolipids induced mild lesions. The small-sized TDM of Rhodococcus possessed granulomatogenic activity, however, the toxicity was much lower than that of M. tuberculosis. Rhodococcal TDM was composed of mycolic acid with the shortest carbon chains, when compared to granulomatogenic TDM of Mycobacterium, Nocardia and Rhodococcus reported previously. Our results imply that rhodococcal TDM is a pathogenetic factor similar to that of M. tuberculosis, although rhodococcal TDM exhibits low toxicity.

Malignant schwannoma of the esophagus with lymph node metastasis: literature review of schwannoma of the esophagus.

An extremely rare case of malignant schwannoma of the esophagus with lymph node metastasis is reported. A 49-year-old woman was found to have an abnormal shadow on a chest X-ray film taken during an annual checkup. Upper gastrointestinal series showed extrinsic pressure on the middle thoracic esophagus, without a mucosal lesion. An exploratory operation was performed, with a tentative diagnosis of esophageal leiomyoma. The tumor was enucleated with part of the esophageal mucosa, and a few enlarged lymph nodes around the tumor were dissected. The resected tumor was an elastic firm mass, measuring 8.2 x 5.8 x 3.7 cm, and had a smooth surface. Histological examination of the tumor revealed the proliferation of spindle-shaped cells with chromatin-rich nuclei. The nuclei were variable in size and showed remarkable atypia. A paraesophageal lymph node had same findings as the main tumor. Immunohistochemically, the tumor cells were diffusely positive for S-100 protein and neuron-specific enolase. The pathological diagnosis of this tumor was malignant esophageal schwannoma with lymph node metastasis. Esophageal schwannoma is extremely rare. We reviewed the literature on 19 cases of esophageal schwannoma, including that in our patient. The majority of the tumors were benign. Only three cases of schwannoma were malignant, and this is the first reported case of malignant schwannoma with lymph node metastasis.

A butter diet induces higher levels of n-3 PUFA and of n-3/n-6 PUFA ratio in rat serum and hearts than a safflower oil diet.

The effects of a 47-week diet of butter or safflower oil as fat in combination with casein or soy protein as protein were observed for the serum concentrations of lipids and fatty acid compositions in rat serum and heart. Serum total cholesterol (Chol) did not differ among the four experimental diet groups. In the butter groups, significantly higher low-density lipoprotein (LDL)-Chol and lower high-density lipoprotein (HDL)-Chol were observed than in the safflower oil groups (p<0.005, respectively). Higher levels of α-tocopherol were found in the butter groups than in the safflower oil groups (p<0.05) and in the casein groups than in the soy protein groups (p<0.01). In comparison with the safflower oil groups, the butter groups showed higher n-3 polyunsaturated fatty acids (PUFA) contents and lower n-6 PUFA contents in serum and the hearts (p<0.005). The ratios of n-3/n-6 PUFA in the butter groups in serum, 0.26 and 0.18, and in the hearts, 0.37 and 0.36, (butter-casein diet and butter-soy protein diet, respectively) were higher than those of the safflower oil groups of under 0.01 in serum and 0.02 and 0.03 in the hearts (safflower oil-casein diet and safflower oil-soy protein diet, respectively) (p<0.005). In the soy protein groups, higher n-3 PUFA contents in the hearts were found than those of the casein groups (p<0.05). This study suggested that the butter diet induces higher levels of n-3 PUFA and a higher n-3/n-6 PUFA ratio than the safflower oil diet in rat serum and hearts over a long feeding period.

Inhibition of 12(S)-hydroxyeicosatetraenoic acid (12-HETE) production suppressed the intimal hyperplasia caused by poor-runoff conditions in the rabbit autologous vein grafts.

The efficacy of OPC-29030, a newly developed inhibitor of 12(S)-hydroxyeicosatetraenoic acid (12-HETE) production, was evaluated on intimal hyperplasia of experimental autologous vein grafts in a distal poor-runoff model and a hyperlipidemic model in rabbits. First, rabbits were divided into two groups, the distal poor-runoff group (PR group) and the hyperlipidemic group (HL group). After 4 weeks preparing the PR model and the HL model, the femoral vein was implanted into the ipsilateral femoral artery. Then they were subdivided into two groups, depending on the diet provided; diet group with 0.1% OPC-29030 (OPC-29030 group) and normal diet group (control group). At 4 weeks, the grafts were harvested, and intimal hyperplasia of the graft was measured with an ocular cytometer. Intimal cell proliferation was determined by bromodeoxyuridine (BrdU) incorporation at 2 weeks after surgery. In addition, the effect of OPC-29030 on the proliferation or migration of rat aortic smooth muscle cells in culture was investigated. In the in vivo study in the PR group, the intimal hyperplasia and the plasma 12-HETE levels in the OPC-29030 group were significantly inhibited, compared with those of the control group. However, in the HL group, the intimal hyperplasia in both the OPC-29030 and control groups showed a remarkable degree of intimal hyperplasia. There was no significant difference between those two groups. Furthermore, there was no significant difference in the plasma 12-HETE levels in the HL group irrespective of the presence of OPC-29030. The BrdU labeling index at 2 weeks after grafting was significantly lower in the OPC-29030 group compared with that in the control group in the PR group. In the in vitro study, OPC-29030 did not inhibit smooth muscle cell proliferation; however, OPC-29030 inhibited the migration. These results demonstrate the efficacy of OPC-29030 in reducing the degree of intimal hyperplasia under PR conditions, but not under hyperlipidemic conditions. The mechanism of reducing the intimal hyperplasia may be that OPC-29030 inhibited 12-HETE production, which did not inhibit proliferation while inhibiting migration of the smooth muscle cell. These results suggested the possible involvement of 12-HETE with the intimal hyperplasia under PR conditions.

Ribozyme approach to downregulate vascular endothelial growth factor (VEGF) 189 expression in non-small cell lung cancer (NSCLC).

The aim of this study was to further clarify the role of the cell-associated isoform of vascular endothelial growth factor (VEGF189) on tumour growth and vascularity. Five isoforms of VEGF have been identified with different biological activities. VEGF121, VEGF145, VEGF165, VEGF189, VEGF206 are generated by alternative splicing. We used a hammerhead-type ribozyme (V189Rz) to suppress VEGF189 mRNA. The V189Rz specifically cleaved exon 6 of VEGF189 mRNA, but showed no activity against the VEGF121 or VEGF165 isoforms. The V189Rz was introduced into the human non-small cell lung cancer (NSCLC) cell line (OZ-6/VR). The expression level of VEGF189 mRNA was decreased in the OZ-6/VR cells, while VEGF121 and 165 expression was unaltered. The OZ-6/VR cells xenotransplanted into nude mice showed markedly reduced vascularisation and growth, whereas the cell line did not show any decreased growth under tissue culture conditions. The OZ-6/VR cells (1 x 10(5) cells/mouse) formed no tumours, whereas the parental OZ-6 cells formed large tumours within 8 weeks. The specific suppression of VEGF189 by the ribozyme decreased vascularity and xenotransplantability of the lung cancer cell line. Thus, the cell-associated isoform of VEGF, VEGF189, might have a key role in stromal vascularisation and the growth of NSCLC xenografts in vivo.