Efficacy of epidermal growth factor receptor-targeted molecular therapy in anaplastic thyroid cancer cell lines.

Anaplastic thyroid cancer is one of the most aggressive human malignancies and the outcomes of conventional therapy have been far from satisfactory. Recently, epidermal growth factor receptor (EGFR)-targeted therapy has been introduced as an alternative therapeutic strategy for highly malignant cancers. This study was undertaken to investigate the expression of EGFR in anaplastic thyroid cancer cell lines, and to explore the potential of therapies targeting EGFR as a new therapeutic approach. EGFR was universally expressed in anaplastic cancer cell lines at a variety of levels. Specific EGFR stimulation with epidermal growth factor showed significant phosphorylation of ERK1/2 and Akt, and resulted in marked growth stimulation in an anaplastic thyroid cancer cell line, which highly expressed EGFR. This EGFR-transmitted proliferation effect of the cancer cell line was completely inhibited by gefitinib, an EGFR tyrosine kinase inhibitor. Moreover, growth of xenografts inoculated in mice was inhibited in a dose-dependent manner with 25-50 mg kg(-1) of gefitinib administrated orally. Inhibition of EGFR-transmitted growth stimulation by gefitinib was clearly observed in anaplastic thyroid cancer cell lines. Our results suggested that EGFR-targeted therapy, such as gefitinib, might be worth further investigation for the treatment of anaplastic thyroid cancer.

Juvenile form of Alexander disease with GFAP mutation and mitochondrial abnormality.

The authors report a 29-year-old woman with marked atrophy of the cerebellum, medulla oblongata, and spinal cord, dementia, diffuse white matter abnormality on MRI, ragged-red fibers, and R88C mutation in the human glial fibrillary acidic protein (GFAP). Mitochondria DNA (mtDNA) analysis showed a rare polymorphism at A8291G. This mtDNA polymorphism, which has been associated with limb-girdle type mitochondrial myopathy, may modify the clinical symptoms of this juvenile form of Alexander disease with GFAP mutation.

Endocrine-disrupting effects of styrene oligomers that migrated from polystyrene containers into food.

The endocrine-disrupting effects of styrene dimers (SD: NSD-01, -08 and -09) and styrene trimers (ST: NST -01, -03 and -12), which migrated from polystyrene (PS) containers into instant food, were investigated together with styrene monomer (SM) using in vitro and in vivo assays. In the estrogen (ER) and androgen receptor (AR) binding assay, SM, SD and ST showed no binding activity at concentration of 10(-10)-10(-5) mol/l. In order to evaluate the estrogenic activity in vivo, the uterotrophic assay was conducted. When prepubertal and ovariectomized adult rats were dosed with SM, SD and ST for 3 days by subcutaneous injection, these compounds did not induce significant increase in uterine weight. Additionally, to evaluate anti-androgen activity in vivo, the Hershberger assay for anti-androgenic activity in the presence of testosterone treatment was conducted. When castrated, testosterone-treated immature male rats were dosed SM, SD and ST for 7 days by oral gavage, these compounds did not induce a decrease in the seminal vesicle, ventral prostate and levator ani plus bulbocavernosus muscle weights. To evaluate the effects on hormones other than sex hormones, the thyroid hormone receptor (TR) binding assay and rat serum prolactin (PRL) was conducted. In the TR binding assay, SM, SD and ST showed no binding activity at a concentration of 10(-5) mol/l. When ovariectomized rats were dosed with SM, SD and ST for 3 days by sc injection, the results showed there was no change in rat serum PRL. From the above these results, we concluded that SM, SD and ST exhibit no apparent estrogenic, androgenic, anti-androgenic and thyroid activity.

Assessment of styrene oligomers eluted from polystyrene-made food containers for estrogenic effects in in vitro assays.

Recently, several substances from among the huge numbers of chemicals used by mankind have been implicated as instigators of disrupted endocrine function and related human health problems. Polystyrene (PS) is one of the most frequently used resins in the world, and the styrene oligomer dissolved out from PS has been designated as a potential trigger of estrogen-like activity in the Wingspread Declaration and the Japan Environment Agency's SPEED98 [JEA (Japan Environment Agency) Strategic Problem on Environmental Endocrine Disruptors '98 (SPEED) '98), http://www.env.go.jp/en/pol/speed98/sp98.html]. In order to assess the endocrine disrupting effect of styrene oligomers, we tested one styrene monomer (SM), three styrene dimers (SDs) and seven styrene trimers (STs), newly isolated from optical isomers, known to dissolve in small amounts from cup noodle containers made of polystyrene by the estrogen receptor binding assay, luciferase reporter gene assay, and human breast cancer cell MCF-7 proliferation assay. In all three tests, none of the SM, SDs and STs showed any significant activity. Accordingly, we concluded that these substances have no estrogenic activity.

Identification, determination, and biological evaluation of a novel styrene trimer contained in polystyrene food containers. 2.

An unidentified styrene trimer (ST) isolated from the acetonitrile extract of polystyrene (PS) food containers was characterized as (1S*,6R*,7S*,8S*,11R*)-6,11-diphenyltricyclo[6,2,2,0(2,7)]dodeca-2,9-diene. The content and migration of this compound in PS food containers were determined by GC-MS (SIM). Furthermore, an endocrine-disrupting effect was tested using in vitro and in vivo assays of the compound. In conclusion, it seems that the compound does not present the effect.

NTE-122, an acyl-coa:cholesterol acyltransferase inhibitor, prevents the progression of atherogenesis in cholesterol-fed rabbits.

The cholesterol-lowering and anti-atherosclerotic effects of NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylaminophenyl)ureido]methyl]cyclohexane), an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, were evaluated in 1% cholesterol diet-fed rabbits. NTE-122 (1, 3 and 10 mg/kg per day) lowered the total cholesterol levels in both plasma and liver dose-dependently (by 99% and 94% at 10 mg/kg per day, respectively). In the aortic wall of the rabbits given NTE-122, the atherosclerotic lesion area in both aortic arch and thoracic aorta were dose-dependently reduced (by 100% at 10 mg/kg per day), and the total cholesterol content in aortic arch was also lowered significantly at more than 3 mg/kg per day. These results suggest that NTE-122 is capable of exhibiting anti-atherosclerotic effects.

Hereditary motor and sensory neuropathy with myelin folding and juvenile onset glaucoma.

OBJECTIVE: We describe three patients from a family with motor and sensory neuropathy accompanied by open-angle glaucoma. BACKGROUND: Autosomal recessive demyelinating hereditary motor and sensory neuropathies (HMSN) include different disorders. To our knowledge, autosomal recessive HMSN has not been associated with juvenile onset glaucoma. METHODS: Sural nerve pathology of the three patients were examined, and genetic analysis of the family was performed. RESULT: - The most prominent pathologic finding was a highly unusual myelin abnormality consisting of irregular redundant loops and folding of the myelin sheath. The family survey supports autosomal recessive inheritance. The molecular analysis failed to demonstrate either linkage of the disease to MPZ gene, PMP22 gene, Cx32 gene, orEGR2 gene. Analysis did not establish linkage of the disease to the locus of CMT4A, 4B, and 4C genes. CONCLUSION: The present cases may represent a new type of HMSN accompanied by juvenile onset glaucoma.

HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) with amyotrophic lateral sclerosis-like manifestations.

To clarify the existence of HAM/TSP presenting amyotrophic lateral sclerosis (ALS)-like manifestations, we assayed HTLV-I proviral load in peripheral blood mononuclear cells (PBMC) in 15 patients with anti-HTLV-I antibody in serum and ALS-like manifestations (upper motor neuron involvement in at least one region and lower motor neuron involvement in at least two limbs) by quantitative PCR, and compared the proviral load with that of 233 HAM/TSP patients and of 213 HTLV-I carriers. Five of 15 patients with ALS-like manifestations had proviral loads as high as those in the 233 patients with HAM/TSP. Anti-HTLV-I antibody in cerebrospinal fluid (CSF) was present in all of five patients. The proviral load in the remaining 10 patients was similar to that in HTLV-I carriers. Four of five patients with a high proviral load met the diagnostic criterion of HAM/TSP except for lower motor neuron involvement. These four patients showed high neopterin levels in CSF. On the basis of HTLV-I proviral load in PBMC and the clinical symptoms, our tentative conclusion is that these four patients are HAM/TSP presenting ALS-like manifestations.

Effects of NTE-122, an acyl-CoA:cholesterol acyltransferase inhibitor, on cholesterol esterification and lipid secretion from CaCo-2 cells, and cholesterol absorption in rats.

The effect of NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl)ureido]methyl]cyclohexane), an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on cholesterol absorption was investigated. NTE-122 inhibited whole-cell ACAT activity in CaCo-2 cells, a human intestinal cell line, with an IC50 value of 4.7 nM. In CaCo-2 cells cultured on a membrane filter, NTE-122 pronouncedly inhibited the basolateral secretion of newly synthesized cholesteryl esters, and significantly reduced the basolateral secretion of newly synthesized triglycerides without influencing the cellular triglyceride synthesis. Furthermore, NTE-122 (1 mg/kg, p.o.) inhibited [14C]cholesterol absorption in rats. These results suggest that NTE-122 is capable of exhibiting anti-hyperlipidemic effects by reducing the absorption of dietary cholesterol.

Effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase inhibitor, on cholesterol esterification and secretions of apolipoprotein B-containing lipoprotein and bile acids in HepG2.

We studied the effect of NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl) ureido]methyl]cyclohexane), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on intracellular cholesterol esterification and the secretion of apolipoprotein B100 (apoB)-containing lipoprotein and bile acids in the human hepatoma cell line HepG2. NTE-122 markably inhibited [3H]oleate incorporation into cholesteryl esters in HepG2 cells incubated with 5 microg/ml 25-hydroxycholesterol as a stimulus for ACAT (IC50=6.0 nM). On the other hand, NTE-122 did not affect [3H]oleate incorporation into triglycerides and phospholipids and [14C]acetate incorporation into cholesterol. The stimulation of ACAT by 25-hydroxycholesterol caused significant increases in the secretion of radiolabeled cholesteryl esters, radiolabeled triglycerides and apoB mass. NTE-122 pronouncedly inhibited the secretion of radiolabeled cholesteryl esters in proportion to the inhibition of cellular cholesterol esterification, and it significantly reduced the secretion of radiolabeled triglycerides and apoB mass in HepG2 cells incubated with 25-hydroxycholesterol. Furthermore, NTE-122 increased the secretion of bile acids synthesized from [14C]-cholesterol. These results suggest that NTE-122 is capable of exhibiting anti-hyperlipidemic effects by reducing both the cholesterol content and the amount of secreted very low-density lipoprotein and enhancing the excretion of bile acid from the liver.

Effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase inhibitor, on cholesterol esterification and high-density lipoprotein-induced cholesterol efflux in macrophages.

We investigated the effects of a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl)ureido]methyl]cyclohexane), on ACAT activities in macrophages originating from several species and high-density lipoprotein (HDL)-induced cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells. NTE-122 inhibited cell-free ACAT activities in human PMA-treated THP-1 cells and mouse J774.1 cells with IC50 values of 0.88 and 360 nM, respectively. NTE-122 competively inhibited the ACAT activity in PMA-treated THP-1 cells. NTE-122 also inhibited cellular ACAT activities in PMA-treated THP-1 cells, rat peritoneal macrophages and J774.1 cells with IC50 values of 3.5, 84 and 6800 nM, respectively. Furthermore, NTE-122 prevented cholesterol accumulation in PMA-treated THP-1 cells incubated with acetylated low density lipoprotein, simultaneously with HDL, while it caused accumulation of a significant amount of free cholesterol in the absence and even in the presence of HDL. NTE-122 also enhanced HDL-induced cholesterol efflux from established foam cells converted from PMA-treated THP-1 cells. These results suggest that NTE-122, capable of inhibiting macrophage ACAT activity in humans more strongly than those in the other species, exhibits anti-atherogenic effects by preventing the foam cell formation and enhancing the foam cell regression in humans.

Manganese intoxication during total parenteral nutrition: report of two cases and review of the literature.

We report two cases of manganese (Mn) intoxication during total parenteral nutrition including manganese (Mn). Both patients showed parkinsonism with psychiatric symptoms and elevated serum Mn levels. T1-weighted magnetic resonance images (MRI) revealed symmetrical high intensity lesions in the globus pallidus. Discontinuation of Mn supplementation and levodopa treatment improved the symptoms and MRI abnormalities in the both patients. Thus, careful attention should be paid to the long-term intravenous administration of Mn.

Cholesterol-lowering effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on cholesterol diet-fed rats and rabbits.

Pharmacological characterization of NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl)ureido]methyl]cyclohexane), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was performed with both in vitro and in vivo assay systems. NTE-122 inhibited microsomal ACAT activities of various tissues (liver of rabbit and rat, small intestine of rabbit and rat, and aorta of rabbit) and cultured cells (HepG2 and CaCo-2), with IC50 values from 1.2 to 9.6 nM. The inhibition mode of NTE-122 was competitive for HepG2 ACAT. NTE-122 had no effect on other lipid metabolizing enzymes, such as 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA synthetase, cholesterol esterase, lecithin:cholesterol acyltransferase, acyl-CoA:sn-glycerol-3-phosphate acyltransferase and cholesterol 7alpha-hydroxylase up to 10 microM. When NTE-122 was administered to the cholesterol diet-fed rats, serum and liver cholesterol levels were markedly reduced with an ED50 of 0.12 and 0.44 mg/kg/day, respectively. In the cholesterol diet-fed rabbits, NTE-122 significantly lowered plasma and liver cholesterol levels at more than 2 mg/kg/day. These results indicate that NTE-122 is a potent, selective and competitive inhibitor of ACAT, making it a worth while therapeutic agent for hypercholesterolemia and atherosclerosis.

Cytoprotective effects of 4,6-bis(1H-pyrazol-1-yl)pyrimidine and related compounds on HCI.ethanol-induced gastric lesions in rats.

Bis(1H-pyrazol-1-yl)- and bis(1H-imidazol-1-yl)pyrimidines were synthesized and evaluated for cytoprotective effects. Among them, 4,6-bis(1H-pyrazol-1-yl)pyrimidine (3) showed a potent inhibitory effect on the HCl.ethanol-, ethanol-, and water immersion stress-induced gastric lesions in rats, and a very low acute toxicity. One of the major factors responsible for the cytoprotective effects of 3 is the increase in the bicarbonate secretion. This compound appears to be a promising cytoprotective drug for the treatment of gastric mucosal ulcers.

Photodegradation products of a new antibacterial fluoroquinolone derivative, orbifloxacin, in aqueous solution.

A new antibacterial fluoroquinolone derivative, orbifloxacin (ORFX), is decomposed photochemically in aqueous solution. When ORFX solution was irradiated with a chemical lamp or sunlight, three major photodegradation products were isolated by preparative HPLC. These degradation products were identified by electron-impact mass spectrometry, liquid-secondary-ion mass spectrometry and 1-H-NMR spectroscopy. Moreover, the photodegradation pathway was investigated by a similar study using several fluoroquinolone derivatives which were presumed to be the intermediates of the photoreaction of the ORFX. Consequently, it was found that two main photochemical reactions, the decomposition of the dimethylpiperazinyl moiety and the elimination of the cyclopropyl group, take place in ORFX. The detected structures of photodegradation products and the photodegradation studies of the postulated intermediates suggested that the photodecomposition of the dimethylpiperazinyl ring at the 7-position and the elimination of the cyclopropyl group at the 1-position occurred concurrently with the release of fluorine at the 8-position.

Synthesis and cytoprotective antiulcer activity of 2- or 4-(1H-pyrazol-1-yl)pyrimidine derivatives related to mepirizole and dulcerozine.

(1H-Pyrazol-1-yl)-, (1H-imidazol-1-yl)-, and (1H-1,2,4-triazol-1-yl)pyrimidines were prepared and evaluated for cytoprotective antiulcer activity. Among them, 4-methoxy-6-methyl-2-(1H-pyrazol-1-yl)pyrimidine (18) showed potent inhibition of the HCl-ethanol-induced and water-immersion stress-induced ulcers in rats, as well as low acute toxicity.

Photodegradation kinetics of the new antibacterial fluoroquinolone derivative, orbifloxacin, in aqueous solution.

The photodegradation kinetics of orbifloxacin (1-cyclopropyl-5,6,8-trifluoro-1,4-dihydro-7-(cis-3,5-dimethyl-1-pipe raz inyl)-4-oxoquinoline-3-carboxylic acid) was investigated in aqueous solution at various pH values (1.2-12.5) and at an ionic strength of 0.5. The photodegradation experiments were performed using a fluorescent or a chemical lamp as a light source and the cumulative number of photons during exposure was determined by a ferrioxalate actinometer. It was found that the photodegradation of orbifloxacin followed apparent first-order kinetics under both types of artificial light. The photodegradation rates of orbifloxacin in a neutral medium were higher than those in acidic and alkaline media. Orbifloxacin was most unstable in solution at pH 7.4, and its degradation half-life was 0.9 h. Also, the log k-pH profile indicated that the photodegradation rate of orbifloxacin was related to the dissociation of the carboxylic and dimethylpiperazinyl groups and the main photo-labile species was the zwitterionic form. In addition, the photodegradation kinetics of the decarboxylated derivative of orbifloxacin in aqueous solution was investigated to determine the effect of the functional groups on the photodegradation of orbifloxacin.

Degradation kinetics of the new antibacterial fluoroquinolone derivative, orbifloxacin, in aqueous solution.

The degradation kinetics of orbifloxacin [1-cyclopropyl-5,6,8-trifluoro-1,4-dihydro-7-(cis-3,5-dimethyl-1-pipe raz inyl)-4-oxoquinoline-3-carboxylic acid] was investigated as a function of pH (1.5-10.5), temperature (100-120 degrees C) and buffer concentration (0.05-0.2 M) by means of high-performance liquid chromatography. The degradation of orbifloxacin in aqueous solution followed apparent first-order kinetics under all experimental conditions. No appreciable effect of buffer on the degradation of orbifloxacin was observed for any of the buffer species used in this study. The log k-pH profiles indicated specific-acid and specific-base catalyses and there were inflection points near pH 6 and 9 corresponding to the pKa1 and pKa2 values. From the Arrhenius plots, the activation energies for k'H, k'H2O, kH2O, k"H2O and k"OH were found to be 31.9, 36.9, 23.5, 26.5 and 19.0 kcal/mol, respectively. Arrhenius data obtained from this study showed that the degradation of orbifloxacin at room temperature was negligible at all pH values studied conditions (pH 1.5-10.5).

Effects of hemorrhagic shock on alkaline secretion and mucosal tolerance to acid in rat duodenum. A comparative study with indomethacin.

The effects of hemorrhagic shock (HE) on duodenal HCO3- secretion and mucosal tolerance to acid were investigated in anesthetized rats and compared with those of indomethacin. HE was performed by bleeding from the carotid artery to reduce arterial blood pressure to about 50 mm Hg (3 ml bleeding per 200 g of body weight) with a significant decrease in arterial pH and [HCO3-], and indomethacin was given subcutaneously in a dose of 5 mg/kg. The proximal duodenum (1.7 cm) secreted HCO3- at the rate of 1.5-1.8 mueq/15 min (3.5-4.2 mueq/cm/hr), and responded to luminal acid (10 mM HCl for 10 min) by a significant rise in HCO3- output. Indomethacin had no effect on basal HCO3- output but significantly inhibited the acid-induced HCO3- secretion, while under HE conditions duodenal HCO3- output significantly declined and failed to increase in response to luminal acidification. Subcutaneously administered 16,16-dmPGE2 (30 micrograms/kg) significantly increased HCO3- secretion in the presence of indomethacin but had less effect on the impaired HCO3- output caused by HE. In contrast, intravenous infusion of NaHCO3 (3 mmol/kg/hr) ameliorated the acid-base imbalance caused by HE, and significantly restored the impaired HCO3- responses induced by HE but not by indomethacin. Both HE and indomethacin induced extensive damage in the mucosa when the duodenal loop was perfused with 50 mM HCl for 1.5 hr, and these lesions were significantly reduced by NaHCO3 infusion and 16,16-dmPGE2, respectively. These results suggest that HE impaired duodenal HCO3- secretion and reduced the tolerance of the mucosa to acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional and morphological alterations in the rat stomach following exposure to hypertonic NaCl solution.

We examined the effects of 1 M NaCl as a mild irritant on gastric potential difference (PD), acid secretion, mucosal blood flow (MBF), and DNA synthetic activity in anesthetized rat stomachs and compared these effects with those of 4 M NaCl as a strong irritant. Both 1 M and 4 M NaCl produced a PD reduction (mucosal injury), but the reduced PD recovered faster in the mucosa exposed to 1 M NaCl as compared to 4 M NaCl. Acid secretion ceased after exposure to these hypertonic NaCl solutions, but histamine infusion (8 mg/kg/hr) stimulated acid secretion only in the mucosa exposed to 1 M NaCl. The MBF was significantly increased in response to 1 M NaCl, while exposure to 4 M NaCl had no effect on the MBF. These changes in acid secretion and MBF induced by 1 M NaCl were significantly antagonized by pretreatment with indomethacin (5 mg/kg, s.c.). The levels of PGE2 and 6-keto PGF1 alpha in the corpus mucosa were significantly increased in the stomach exposed to both 1 M and 4 M NaCl, and these increases disappeared in the presence of indomethacin. The rate of [3H]-thymidine incorporation was significantly reduced in the mucosa after exposure to 4 M NaCl, but remained unaltered in the stomach exposed to 1 M NaCl. These results suggest that although both 1 M and 4 M NaCl produced mucosal injury (PD reduction) and enhanced PGs formation, a variety of functional alterations mediated by PGs occurred in response to injury in the stomach exposed to 1 M NaCl. The presence or absence of these functional responses may be associated with the biphasic actions on the gastric mucosa of these hypertonic NaCl solutions as mild and strong irritants.