Genomic organization, expression, and chromosome location of the human SNAIL gene (SNAI1) and a related processed pseudogene (SNAI1P).

Some of the zinc finger proteins of the snail family are essential in the formation of mesoderm during gastrulation and the development of neural crest and its derivatives. We have isolated the human SNAIL gene (HGMW-approved symbol SNAI1) and describe its genomic organization, having sequenced a region spanning more than 5882 bp. The human SNAIL gene contains three exons. The SNAIL transcript is 2. 0 kb and is found in placenta and adult heart, lung, brain, liver, and skeletal muscle. It codes for a protein of 264 amino acids and 29.1 kDa. This protein contains three classic zinc fingers and one atypical zinc finger. The human SNAIL protein is 87.5, 58.7, 50.9, 50.7, 55.4, and 31.5% identical to mouse Snail, chicken snail-like, zebrafish snail1, zebrafish snail2, Xenopus snail, and Drosophila snail proteins, respectively. The zinc finger region is 95.5% identical between human and mouse Snail. Because Drosophila snail and twist are important regulators during mesoderm development and because human TWIST mutations have been implicated in craniosynostosis, a cohort of 59 patients with craniosynostosis syndromes were screened for SNAIL mutations. None were found. By somatic cell and radiation hybrid mapping panels, SNAIL was localized to human chromosome 20q13.2, between markers D20S886 and D20S109. A SNAIL-related, putative processed pseudogene (HGMW-approved symbol SNAI1P) was also isolated and maps to human chromosome 2q33-q37.

Radiosensitization of two murine tumours with mild temperature hyperthermia and carbogen breathing.

PURPOSE: To test the individual and combined effects of local mild temperature hyperthermia (MTH) at 41.5 degrees C for 60 min and carbogen breathing on tumour radiosensitivity. MATERIALS AND METHODS: The FSall fibrosarcoma of C3H mice and the SCK mammary carcinoma of A/J mice were used. The effect of various treatments on tumour cell survival was determined using the in vivo/in vitro tumour excision assay, and the radiobiological hypoxic fraction was calculated. The tumour radiation sensitivity was tested with the tumour growth delay assay. RESULTS: The radiobiological hypoxic cell fraction (HF) in control FSall and SCK tumours was 0.45 and 0.78, respectively, and these values decreased to 0.12 in FSall tumours and 0.22 in SCK tumours when determined immediately after the tumours were treated with MTH. The HF was 0.32 in FSall tumours and 0.33 in SCK tumours after carbogen breathing was applied. When tumours were treated with MTH and the animals breathed carbogen the HF decreased to 0.03-0.04 in both FSall and SCK tumours. MTH treatment alone had only a small effect on tumour growth, but MTH treatment applied before irradiating the tumours significantly increased the radiation-induced tumour growth delay. Carbogen breathing modestly improved the radiation-induced tumour growth delay while the combination of MTH treatment and carbogen breathing caused the largest increase in radiation-induced tumour growth delay. CONCLUSIONS: MTH treatment alone and combined with carbogen breathing substantially increased the tumour radiation response probably through an increase in the tumour oxygenation status.

The optimal combination of hyperthermia and carbogen breathing to increase tumor oxygenation and radiation response.

PURPOSE: To determine the most effective combination of carbogen breathing with mild temperature hyperthermia (MTH) to increase the oxygenation and radiation response in murine tumors. METHODS AND MATERIALS: MTH at 41.5 degrees C for 60 min was applied by immersion of the tumor in a precisely controlled water bath. The tumor pO2 was measured with a polarographic microelectrode. The radiation response of the tumor was determined using the in vivo/in vitro assay for surviving tumor cells. RESULTS: In the FSaII fibrosarcoma the median pO2 increased from a control value of 6.5 +/- 0.5 mm Hg to 16.6 +/- 1.1 mm Hg immediately after MTH and was 10.9 +/- 1.3 mm Hg 24 h later. Carbogen breathing for 5 min increased the FSaII pO2 to 19.9 +/- 2.1 mm Hg. Carbogen breathing for 5 min beginning immediately after MTH increased the median pO2 more than 5 times to 35.4 +/- 3.8 mm Hg. This combined treatment also substantially increased the response of the tumors to a radiation exposure of 20 Gy. In another tumor model, the SCK mammary carcinoma, MTH treatment increased the median pO2 from the control level of 4.4 +/- 0.2 mm Hg to 12.6 +/- 1.2 mm Hg, and it returned to 4.3 +/- 0.3 mm Hg 24 h later. Carbogen breathing for 5 min increased the SCK tumor pO2 to 17.1 +/- 1.4 mm Hg. The median SCK pO2 was increased about 7 times to 31.2 +/- 4.2 mm Hg when MTH was followed immediately with carbogen breathing for 5 min. The radiation response was also markedly increased by this combination. When the animals breathed carbogen for 15 or 30 min, the pO2 and radiosensitivity in both tumor types either remained the same or was lower than that after 5 min of breathing. In addition, both FSaII and SCK tumors were radiosensitized 24 h after MTH treatment alone or with 5 min of carbogen breathing. CONCLUSIONS: A shorter carbogen breathing time immediately after MTH causes the most tumor radiosensitization. The results of this study also demonstrate that MTH increases radiosensitivity with and without carbogen breathing up to 24 h after the mild hyperthermia treatment.

Tumor oxygenation after mild-temperature hyperthermia in combination with carbogen breathing: dependence on heat dose and tumor type.

We investigated the effect of hyperthermia at 40.5-42.5 degrees C as well as the combination of hyperthermia and carbogen breathing on oxygenation in the SCK murine mammary carcinoma. In addition, the important question of how long the effect of heating on tumor oxygenation lasts was addressed in both SCK and FSaII (murine fibrosarcoma) tumors. The median pO2 in control SCK tumors was 4.4 +/- 0.2 mm Hg, and it increased to a maximum of 12.6 +/- 1.2 mm Hg when the tumors were heated at 41.5 degrees C for 1 h. Carbogen breathing increased the median pO2 of SCK tumors to 17.1 +/- 1.4 mm Hg, but after heating at 41.5 degrees C, it elevated the pO2 in SCK tumors markedly to 31.3 +/- 4.2 mm Hg. The kinetics of the return to baseline oxygenation after hyperthermia was found to vary with the type of tumor and the heat dose. The pO2 of FSaII tumors remained significantly higher than that of control tumors 24 h after heating at 41.5 degrees C for 60 min. The pO2 of SCK tumors remained elevated for up to 3 h after heating at 41.5 degrees C for 30 min, but if the tumors were heated for 60 min at this temperature, the median oxygen tension declined to the control level within 1 h after heating. It was concluded that mild-temperature hyperthermia, i.e. 41.5 degrees C, alone and in combination with carbogen breathing dramatically improves the oxygenation of these murine tumors and that the tumor type influences the duration of changes in oxygenation induced by mild-temperature hyperthermia.

Antithrombin III (AT III) prevents LPS-induced pulmonary vascular injury: novel biological activity of AT III.

Acute respiratory distress syndrome (ARDS) adversely affects the outcome of patients with disseminated intravascular coagulation (DIC) associated with sepsis. To determine whether antithrombin III (AT III) is useful for the treatment of ARDS in sepsis, we evaluated the effect of AT III on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. Although the intravenous administration of AT III (250 U/kg) prevented LPS-induced pulmonary accumulation of leukocytes, increases in pulmonary vascular permeability, and coagulation abnormalities, inactivated factor Xa, a selective inhibitor of thrombin generation, did not prevent such events other than the coagulation abnormalities. AT III promotes the endothelial release of prostacyclin by interacting with cell surface glycosaminoglycans in vivo. Trp49-modified AT III, which lacks affinity for heparin, did not prevent LPS-induced pulmonary vascular injury. Plasma levels of 6-keto-prostaglandin F1alpha were markedly increased in rats after the administration of LPS and significantly decreased in the LPS-treated rats administered Trp49-modified AT III, but not altered in those LPS-treated rats receiving AT III. Preventive effects of AT III were not observed in rats pretreated with indomethacin, which inhibits prostacyclin biosynthesis. Prostacyclin prevents LPS-induced pulmonary vascular injury by inhibiting leukocyte accumulation in the lungs. These observations strongly suggest that AT III prevents pulmonary vascular injury induced by LPS by promoting the endothelial release of prostacyclin, a potent inhibitor of leukocyte activation.

Improvement of tumor oxygenation status by mild temperature hyperthermia alone or in combination with carbogen.

The effects of mild temperature hyperthermia (MTH) on the oxygenation and radioresponse in rodent tumors were investigated. FSall tumors grown in the legs of C3H mice and R3230 AC tumors grown in the legs of Fischer rats were heated with a water bath and the partial pressure of oxygen (pO2) was determined using the microelectrode method. In FSall tumors, the pO2 measured immediately after heating at 41.5 degrees C for 1 hour was markedly higher than that in the control tumors, whereas heating at higher temperatures for 1 hour decreased the tumor oxygenation. In R3230 AC tumors, heating at 41.5 degrees C for 1 hour caused a moderate increase in the pO2 and heating at 42.5 degrees or 43.5 degrees C for 30 minutes markedly increased the pO2. However, heating at 42.5 degrees C or higher temperatures for 1 hour decreased the pO2 in the R3230 AC tumors. The improvement of oxygenation in FSall tumors by heating at 41.5 degrees C for 1 hour increased the radiation-induced cell death in these tumors. The combination of carbogen breathing with MTH was far more potent than carbogen breathing or MTH alone in increasing tumor oxygenation and potentiating the radiation effect in FSall tumors.

Mild temperature hyperthermia combined with carbogen breathing increases tumor partial pressure of oxygen (pO2) and radiosensitivity.

The partial pressure of oxygen (pO2) of FSaII tumors grown in the leg of C3H mice significantly improved when the tumors were heated by immersing the tumor-bearing legs in a water bath at 41.5 degrees C for 60 min. The tumor pO2 also substantially increased when the tumor-bearing mice breathed carbogen (95% O2:5% CO2). Additionally, mild hyperthermia followed by carbogen breathing further increased the tumor pO2 and increased radiation cytotoxicity as assessed by the in vivo/in vitro excision assay for surviving FSaII cells. It was concluded that mild hyperthermia in combination with carbogen breathing is potentially useful to reoxygenate radioresistant hypoxic cells and improve the radiotherapy of human tumors.

Protective effects of antithrombin III supplementation on warm ischemia and reperfusion injury in rat liver.

The effect of antithrombin III (AT III) supplementation on energy status, microcirculation, cytoprotection, and prostacyclin (PGI2) production during and after a period of warm ischemia of the rat liver was investigated. AT III supplementation (250 units/kg) stimulate prostaglandin I2 (PGI2) production from 1 hour after administration, with maximal production observed at 3 hours. Ischemia was induced by occluding the hepatoduodenal ligament for 30 minutes, and experiments were continued for 60 minutes after reperfusion. The rats received AT III (250 units/kg IC) 30 minutes before induction of liver ischemia (AT III group). In the AT III group, recovery of the beta-ATP/inorganic phosphate ratio measured by 31P nuclear magnetic resonance showed significant improvement (p < 0.01), and the recovery of tissue blood flow markedly improved (p < 0.01) compared to the saline-treated group (control group). Leakages of aspartame aminotransferase, alanine aminotransferase, and lactate dehydrogenase were mitigated in the AT III group (p < 0. 05). Ultrastructural alterations of sinusoidal endothelial cells were markedly reduced in the AT III group. The PGI2 level at the end of reperfusion was significantly elevated (p < 0.01) in the AT III group compared to the control group. The results of this study indicated that pretreatment with AT III significantly improved the energy status and microcirculation, as well as histologic damage, after liver ischemia and reperfusion. One of the fundamental effects of AT III might be mediated through the production of prostacyclin.

[A possible mechanism of interstitial pneumonia during interferon therapy with sho-saiko-to].

Interstitial pneumonia has been reported to be a side effect of treatment with interferon, and Sho-saiko-to (Xiao-Chai-Hu-Tang) may enhance this side effect. It is well known that activated neutrophils are important mediators of pulmonary fibrosis, so we studied the effects of interferon and Sho-saiko-to on neutrophil activation. Homogenized lung myeloperoxidase (MPO) activity was assayed after intraperitoneal injection of interferon with or without pretreatment with Sho-saiko-to. Although Sho-saiko-to alone did not change the lung MPO content, MPO in the lung was significantly increased by interferon administration. The increase was enhanced further by pretreatment with Sho-saiko-to. When the accumulated neutrophils are activated by some cytokines, such as TNF alpha or IL-1 beta from monocytes/macrophages, they may damage lung tissue. We therefore studied the effects of Sho-saiko-to and interferon on TNF alpha production in freshly isolated human monocytes. Sho-saiko-to increased the production of TNF alpha, but interferon did not. In addition, Sho-saiko-to significantly increased the production of TNF alpha by monocytes stimulated by lipopolysaccharide. Taken together, these data indicate that interferon causes neutrophils to accumulate in the lung. Sho-saiko-to alone may not injure lung tissue, but it increases the effect of interferon. When stimulated by some antigen, Sho-saiko-to may overstimulate the neutrophils. Granulocytes elastase and oxygen radicals released from activated neutrophils may damage lung tissue. The fibroblasts that repair the damaged tissue may increase the risk of pulmonary fibrosis.

Development of intra-arterial hyperthermia using a dextran-magnetite complex.

Dextran-magnetite complex (DM) is a colloidal sol of subdomain magnetite particles (i.e. a 'magnetic fluid'). The specific absorption rate of DM in an AC magnetic field is much higher than those of multidomain ferrite particles due to its different mechanism of heat generation. We designed two DM-containing embolic materials (a DM/Lipiodol emulsion and a DM/degradable starch microsphere suspension) to heat target tissues with the use of an external AC magnetic field. In vitro experiments showed that the heat-generating capacity of DM was not reduced in these mixtures. When these materials were injected into the renal arteries of Japanese white rabbits, they both achieved embolization of the artery and selective heating of the embolized kidney by exposure to a 100 kHz AC magnetic field of approximately 15,000 A/m. Histological examination showed a homogeneous distribution of DM in the embolized kidney. These results suggest the possibility of using DM-containing embolic materials for inductive hyperthermia.

[Overview of clinical trials on adjuvant chemotherapy for curatively resected gastric cancer].

Reports of the results of clinical trials on adjuvant chemotherapy for curatively resected gastric cancer were reviewed. All but two trials did not show its efficacy. In spite of this, understanding some results of subset analysis as definite proof, a consensus was achieved in Japan on its efficacy due to lack of proper knowledge of medical statistics among surgeons. Recently it has been recognized that this consensus was groundless and that evaluation of adjuvant chemotherapy should be carried out again. The results of the trial of JCOG, comparing adjuvant chemotherapy versus control (surgery alone) are therefore awaited. For the moment, all clinical trials on adjuvant chemotherapy for curatively resected gastric cancer should have an arm of surgery alone as control.

Intraarterial infusion of autologous lymphocytes for the treatment of refractory lymphoedema. Preliminary report.

OBJECTIVE: To evaluate the results of the treatment of lymphoedema by intra-arterial infusion of autologous lymphocytes. DESIGN: Open study. SETTING: University Hospital. SUBJECTS: 13 patients with refractory lymphoedema. INTERVENTIONS: Lymphocytes were separated from the patient's own blood using a blood cell separator; about 100 cc of lymphocyte dominant blood separated from this blood was immediately infused into the proximal artery of the affected limb. Infusion was practiced once a week, and repeated 4 to 6 times. MAIN OUTCOME MEASURES: Change in size of the affected limb (defined as the difference between the affected limb and the normal limb after treatment), and softening of the edema (measured with a tension gauge). RESULTS: In all 13 patients there was softening of the affected hard limb followed by a reduction in the size of the limb (mean 64%), and the ache and sensation of heat in the limb lessened. The reduction in size was maintained in 9 of the 13 patients for three months, despite returning to their normal activities. CONCLUSION: Intra-arterial infusion of autologous lymphocytes is a promising treatment for refractory lymphoedema.

CT manifestations of a ruptured hepatic tumor after transcatheter arterial embolization.

There are few reports of the radiologic diagnosis of ruptured hepatic tumors. In a patient with right upper abdominal pain and impending shock, angiography demonstrated a hypervascular hepatic tumor, and CT imaged an extrahepatic mass suggestive of a hematoma. Following transcatheter arterial embolization with Lipiodol Ultrafluide and gelatin-sponge, multiple contiguous CT sections revealed numerous lipiodol droplets adjacent to a lipiodol-containing hepatic tumor, clearly outside the liver. These findings were indicative of a ruptured hepatic tumor. After embolization, the patient's condition improved and he was discharged.

[Hepatic arterial infusion chemotherapy of hepatocellular carcinoma].

In 24 cases of unresectable hepatocellular carcinoma, we performed hepatic arterial catheterization and intra-arterial infusion chemotherapy. Adriamycin (ADM), Mitomycin C (MMC), 5-FU and Lipiodol (LPD) were administered an average of 13.5 times over a mean period of 106 days. Except for 5 unevaluable cases, there were 0 CR, 5 PR, 4 MR, 7 NC and 2 PD cases, for an efficiency rate of 27.8%. Complications thought to be due to the catheter included catheter blockade in 1 case (4.3%) and dermal infection of insertion site in 3 cases (13.0%). As for the results of follow-up study, one-year survival rate with this therapy was 47.8%, which compares favorably with a one-year survival rate of 30.0% in 30 cases treated only with TAE. From the above results, hepatic arterial infusion chemotherapy can be repeatedly performed on an outpatient basis, and it is considered to be a useful therapeutic method for treating unresectable hepatocellular carcinoma.

Increase of protein synthesis by uridine supplement in lectin-stimulated peripheral blood lymphocytes and EB virus-transformed B cell line of hereditary orotic aciduria type I.

A 2 month-old Japanese girl with hereditary orotic aciduria type I was treated with oral uridine supplement. The activities of orotate phosphoribosyltransferase (OPRT) and orotidine-5'-phosphate decarboxylase (ODC) in erythrocytes were 2.7 and 0.4%, respectively, of those in the controls. Megaloblastic anemia, excessive urinary excretion of orotic acid, lymphopenia and decreased number of OKT3 positive lymphocytes on admission were corrected after the uridine supplement. Peripheral blood lymphocytes (PBL) were cultured for 24 hr in RPMI 1640 medium with 10% heat-inactivated fetal calf serum and further stimulated with PHA-P, ConA or PWM in the presence of 10 to 1000 microM uridine. EB virus-transformed B cell line (LCL) maintained with an optimal concentration of uridine was cultured for 48 hr in uridine free medium and cultured for an additional 48 hr with 1 to 1000 microM uridine. The incorporations of leucine in to PHA-, ConA- and PWM-stimulated PBL and into LCL of the patient increased in the presence of uridine over 10 microM, although they did not increase in controls. These data suggest that low protein synthesis might correlate with an immune deficiency in hereditary orotic aciduria type I.

Inter-organ metabolism and transport of a cysteine-S-conjugate of xenobiotics in normal and mutant analbuminemic rats.

Biosynthesis of N-acetylcysteine S-conjugates of toxic electrophiles, mercapturic acids, occurs via inter-organ metabolism and transport in which liver, small intestine and kidney play an important role. Since a mercapturic acid is a hydrophobic organic anion and strongly binds to plasma albumin in vitro, the ligand-albumin interaction may affect the metabolic fate of this final metabolite in vivo. To investigate the role of the circulating albumin in detoxication and elimination of a toxic electrophile, urinary occurrence of the final metabolite was determined in normal and mutant Nagase analbuminemic rats (NAR) after administration of S-benzylcysteine, a model compound of cysteine conjugates. S-Benzylcysteine intravenously administered was excreted rapidly into urine as its N-acetyl derivative in both animal groups. However, the urinary recovery of this mercapturic acid was significantly lower in NAR than in normal animals. The lower urinary recovery in NAR was due to a rapid and random distribution of the unbound metabolite in the circulation to extrarenal tissues. In contrast, no significant difference in the urinary recovery of the final metabolite was observed between the two animal groups if S-benzylcysteine was given orally. Kinetic analysis revealed that the major part of the orally administered S-benzylcysteine was transferred to the liver and acetylated predominantly in this organ in both animal groups; the mercapturic acid which was synthesized in the liver can be transferred to the kidney and excreted into urine even in the absence of the circulating albumin. These results indicate that albumin is important for a final elimination of a mercapturic acid when animals were extraorally challenged with a large dose of toxic electrophiles by which the rate of biosynthesis and the plasma level of the amphipathic metabolites were increased.