Involvement of brain oxidation in the cognitive impairment in a triple transgenic mouse model of Alzheimer's disease: noninvasive measurement of the brain redox state by magnetic resonance imaging.

Oxidative stress is considered to be related to the onset and/or progression of Alzheimer's disease (AD), but there is insufficient evidence of its role(s). In this study, we evaluated the relationships between the brain redox state and cognitive function using a triple transgenic mouse model of AD (3 × Tg-AD mouse). One group of 3 × Tg-AD mice started to receive an α-tocopherol-supplemented diet at 2 months of age and another group of 3 × Tg-AD mice was fed a normal diet. The levels of α-tocopherol, reduced glutathione, oxidized glutathione, and lipid peroxidation were decreased in the cerebral cortex and hippocampus at 4 months of age in the 3 × Tg-AD mice fed a normal diet. These reductions were abrogated by the supplementation of α-tocopherol in the diet. During Morris water maze testing, the 3 × Tg-AD mice did not exhibit cognitive impairment at 4 months of age, but started to show cognitive dysfunction at 6 months of age, and α-tocopherol supplementation suppressed this dysfunction. Magnetic resonance imaging (MRI) using 3-hydroxymethyl-proxyl as a probe showed decreases in the signal intensity in the brains of 3 × Tg-AD mice at 4 months of age, and this reduction was clearly attenuated by α-tocopherol supplementation. Taken together, these findings suggest that oxidative stress can be associated with the cognitive impairment in 3 × Tg-AD mice. Furthermore, MRI might be a powerful tool to noninvasively evaluate the increases in reactive radicals, especially those occurring during the early stages of AD.

Variations in the structure and transcription of the mitochondrial atp and cox genes in wild Solanum species that induce male sterility in eggplant (S. melongena).

In order to determine the molecular basis of cytoplasmic male sterility (CMS) in alloplasmic lines of eggplant, the genomic structures and transcription patterns of mitochondrial ATP synthase subunit (atp) and cytochrome oxidase subunit (cox) genes were studied for wild and cultivated eggplants. Alloplasmic eggplant lines with cytoplasms of wild Solanum species showing either anther indehiscent type of CMS or non-pollen production type of CMS were studied with the cultivated eggplant Solanum melongena, used as a control. Southern hybridization of the mitochondrial genes indicated the difference between the two types of CMS and showed complete identity within each type. The cytoplasmic patterns of all wild species differed from that of the cultivated eggplant. Thus, the cytoplasm of the six wild eggplants and the one cultivated eggplant was classified into three groups. Male sterile plants of both types of CMS showed novel transcription patterns of atp1, whereas a different transcription pattern of cox2 was observed only in the anther indehiscent type. Based on these differences, we determined the DNA sequences of about a 4 kbp segment in the atp1 region. Although the coding and 3' flanking regions were almost identical among the cytoplasms, the 5' flanking region was completely different and novel open reading frames (orfs) were found for each of the CMS types and the cultivated eggplant. The cytoplasm of Solanum kurzii inducing the anther indehiscent type CMS had orf312, and those of Solanum aethiopicum and Solanum grandifolium of non-pollen production type CMS had orf218. The correspondence between the transcription patterns of these orfs and phenotypic expression of male sterility strongly suggests that these orfs are causal genes for each type of CMS.

Clock and ATF4 transcription system regulates drug resistance in human cancer cell lines.

The mechanisms underlying cellular drug resistance have been extensively studied, but little is known about its regulation. We have previously reported that activating transcription factor 4 (ATF4) is upregulated in cisplatin-resistant cells and plays a role in cisplatin resistance. Here, we find out a novel relationship between the circadian transcription factor Clock and drug resistance. Clock drives the periodical expression of many genes that regulate hormone release, cell division, sleep-awake cycle and tumor growth. We demonstrate that ATF4 is a direct target of Clock, and that Clock is overexpressed in cisplatin-resistant cells. Furthermore, Clock expression significantly correlates with cisplatin sensitivity, and that the downregulation of either Clock or ATF4 confers sensitivity of A549 cells to cisplatin and etoposide. Notably, ATF4-overexpressing cells show multidrug resistance and marked elevation of intracellular glutathione. The microarray study reveals that genes for glutathione metabolism are generally downregulated by the knockdown of ATF4 expression. These results suggest that the Clock and ATF4 transcription system might play an important role in multidrug resistance through glutathione-dependent redox system, and also indicate that physiological potentials of Clock-controlled redox system might be important to better understand the oxidative stress-associated disorders including cancer and systemic chronotherapy.

Mutations within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking.

Somatically acquired mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer are associated with significant clinical responses to gefitinib, a tyrosine kinase inhibitor that targets EGFR. We screened the EGFR in 469 resected tumours of patients with lung cancer, which included 322 adenocarcinomas, 102 squamous cell carcinomas, 27 large cell carcinomas, 13 small cell carcinomas, and five other cell types. PCR with a specific condition was performed to identify any deletion in exon 19, while mutant-allele-specific amplification was performed to identify a mutation in codon 858 of exon 21. EGFR mutations were found in 136 cases (42.2%) with adenocarcinoma, in one case with large cell carcinoma, and in one case with pleomorphic carcinoma. An in-frame deletion in exon 19 was found in 62 cases while an L858R mutation was found in 77 cases. In the 322 cases with adenocarcinoma, these mutations were more frequently found in women than in men (P=0.0004), in well differentiated tumours than in poorly differentiated tumours (P=0.0014), and in patients who were never smokers than in patients who were current/former smokers (P<0.0001). The mutation was more frequently observed in patients who smoked

Clinical features and therapeutic outcomes of 65 patients with acromegaly at Tokyo Women's Medical University.

OBJECTIVE: The purpose of this study was to survey the clinical characteristics, complications, and therapeutic outcome in patients with acromegaly. PATIENTS AND METHODS: The clinical features of 65 patients with acromegaly (31 males, 34 females; mean age: 50+/-2 yr.) who were admitted to Tokyo Women's Medical University between 1990 and 1999 were analyzed retrospectively from medical records. RESULTS: The retrospective analysis revealed that the diagnosis of acromegaly was preceded by approximately 8.1+/-1.1 years of signs and symptoms of the disease. Forty-six of the 65 patients (71%) had macroadenomas, 16 (25%) had microadenomas, and the remaining three had empty sella. The rate of biochemical cure or remission was 81% for microadenoma (13/16), 64% for macroadenoma without extrasellar extension (9/14), and 13% for macroadenoma with cavernous sinus extension (2/15). Eighteen (28%) patients had impaired glucose tolerance (IGT) and 32 (49%) had diabetes mellitus (DM). After treatment for acromegaly, glucose metabolism was analyzed again in 38 patients, and it improved in 26 patients with IGT or DM. Twenty-five of 65 patients (38%) had hypertension. Of 26 patients who underwent barium enema or colonoscopy, 10 had colonic polyps and 4 had colon cancer. CONCLUSION: This study suggests that long-term excessive growth hormone (GH) secretion causes many complications. Therefore, awareness of the early symptoms and signs of acromegaly and long-term careful management of complications, along with therapy to reduce serum GH/insulin-like growth factor (IGF)-I levels, are important for patients with acromegaly.

An evaluation of the bioavailability of selenium in high-selenium yeast.

The bioavailability of selenium (Se) in high-Se yeast (SeY) was evaluated by measuring tissue Se accumulation and glutathione peroxidase (GSHPx) activity. For 4 weeks, 4-week-old male Wistar rats were fed a Torula yeast-based Se-deficient diet (basal diet) or a diet supplemented with a graded level (0.04, 0.08, 0.16, and 0.32 microgram/g) of Se as either sodium selenite or SeY, which was obtained from two different sources. Se supplementation did not influence growth, hematological values, or serum biochemical tests. Se contents and GSHPx activities in the liver, serum, and erythrocytes increased gradually with increases of the supplemented Se. At lower Se levels (0.04 and 0.08 microgram/g), selenite produced higher Se deposition and higher GSHPx activities than SeY did, but at a higher Se level (0.32 microgram/g), SeY showed higher measures. Strong correlations were detected between the supplementary Se levels and the tissue Se contents or GSHPX activities when the regression was fitted to this equation: R-Rb = m log X + k, where R represented tissue Se content or GSHPx activity in rats fed the diet supplemented with Se at X level, Rb corresponding mean value in rats fed the basal diet, m slope, and k constant. The bioavailability of Se in SeY, as assessed by slope ratio analysis using selenite as a reference Se, was 135% to 165% in the tissue Se content and 105% to 197% in the GSHPx activities. These results indicate that Se in SeY is more bioavailable than selenite Se, and therefore it is the preferred form for supplementation.

The effects of long-term treatment on left ventricular hypertrophy in patients with essential hypertension: relation to changes in neurohumoral factors.

This study compared the effects of 1 year of monotherapy with a calcium-channel antagonist (nilvadipine; NIL), an angiotensin-converting enzyme (ACE) inhibitor (temocapril; TEM), or a new vasodilator (cadralazine; CAD) on left ventricular (LV) hypertrophy in essential hypertension. Furthermore, to elucidate the mechanism responsible for regression of LV hypertrophy after treatment, LV mass index (LVMI) by echocardiography, plasma renin activity (PRA), aldosterone (PAC), norepinephrine, and atrial natriuretic peptide (ANP) concentration were measured before and after treatment. Thirty-six patients were randomly assigned to the NIL, TEM, or CAD groups. Blood pressure (BP) before treatment was 174 +/- 10/104 +/- 7, 173 +/- 18/103 +/- 8, and 171 +/- 16/103 +/- 7 mm Hg (mean +/- SD) in NIL, TEM, and CAD groups, respectively. BP was lower after treatment with each of the three test drugs than after the placebo period, and there were no differences in BP reduction among three groups. LVMI, in NIL and TEM, was reduced from 129 +/- 48 to 115 +/- 39 g/m2 and from 117 +/- 39 to 88 +/- 20 g/m2 (p < 0.05 and p < 0.01, respectively), whereas, in the CAD group, it was increased (110 +/- 30 to 138 +/- 27 g/m2; p < 0.01). In the CAD group, PAC decreased and ANP increased significantly. The change in LVMI correlated with that in BP for TEM and with that in ANP in all patients. These data indicated that LV volume overload as well as LV pressure overload may contribute to LV hypertrophy and that monotherapy with CAD is not desirable from the point of view of LV mass reduction in essential hypertension.

Production of recombinant human monoclonal antibody using ras-amplified BHK-21 cells in a protein-free medium.

A ras oncogene-amplified recombinant BHK-21 cell line (ras-rBHK-IgG) has been established, and was shown to hyperproduce the recombinant IgG chimeric human monoclonal antibody (hMAb) AE6F4, which recognizes lung cancer cells. We found that the ras-rBHK-IgG cell could be easily cultured in a protein-free ERDF medium supplemented with iron(III) nitrate, hydroxyethyliminodiacetic acid, and non-protein synthetic attachment factor as well as in a serum-free ERDF medium supplemented with insulin, transferrin, ethanolamine, and sodium selenite. The productivity of recombinant hMAb from the cells cultured in dishes at high cell densities was higher in protein-free medium than in serum-containing medium. True high density culture of the ras-rBHK-IgG cells was done in protein-free medium using the Tecnomouse, which is a novel hollow fiber bioreactor system. After culture for 30 days in protein-free culture, a total amount of about 14 mg of the recombinant hMAb AE6F4 was obtained, and was shown to be reactive against lung cancer cells in tissues.

Histological study of iron deposits in selenium-deficient rats.

Previous studies have shown that selenium (Se) deficiency is associated with hematological abnormalities, which may result in an increased distribution of iron in various tissues. This report describes histological studies of the location of excess iron deposits in tissues. Male Wistar rats were fed a Torula yeast-based Se-deficient [Se(-)] or Se-adequate [Se(+); containing 0.1 mg Se/kg as sodium selenite] diet for 8 or 82 weeks. Excised tissues were embedded in either paraffin or epoxy resin. A dramatic increase was observed in iron deposition in the liver and kidneys of rats on the Se(-) diet. Prussian blue-stained sections under the light microscope showed iron deposits in the parenchymal cells and Kupffer cells of liver and in the proximal tubules of kidneys. The liver and kidneys of Se(-) rats had considerably altered morphology: lysosomes were enlarged and contained electron-dense areas. X-Ray microanalysis showed that the areas that corresponded to the lysosomes contained iron. No iron deposits were observed in sections of kidney and liver from rats fed the Se(+) diet. Thus, these studies identified subcellular sites of iron deposition in the liver and kidneys of Se(-) rats. These iron deposits may be an important factor in the pathogenesis of Se deficiency.

Time course of regression of vascular structural changes and its relation to cytosolic free calcium in hypertensives after nilvadipine treatment.

The objective of this study was to assess the regression of vascular structural changes seen in essential hypertension after long-term monotherapy with a calcium antagonist and to clarify the relations to cytosolic free calcium and neurohumoral factors. Blood pressure, minimal vascular resistance (MVR) by strain-gauge plethysmography, cytosolic free calcium in platelets ([Ca2+]i) by Quin 2 method, plasma renin activity (PRA) and plasma aldosterone concentration (PAC), plasma noradrenaline (PNA) and parathyroid hormone (PTH) were measured in 14 essential hypertensives during a placebo period and 2 and 6 months after anti-hypertensive treatment with nilvadipine. Blood pressure decreased from 174 +/- 10/104 +/- 8 mm Hg during the placebo period to 154 +/- 13/93 +/- 14 mm Hg 2 weeks after nilvadipine, and the hypotensive effects were found throughout the 6-month period. Although increased MVR seen in hypertensives did not change after 2 months (from 2.1 +/- 0.7 to 1.9 +/- 0.6 mm Hg/ml/min per 100 ml tissue (PRU), NS), MVR decreased significantly at 6 months (1.6 +/- 0.4, PRU, P < 0.05). Elevated [Ca2+]i seen in hypertensives during the placebo period decreased significantly 2 months after nilvadipine treatment (156 +/- 26 and 140 +/- 27 nM, P < 0.01). The changes in MVR were associated with those in [Ca2+]i 6 months after nilvadipine (r = 0.56, P < 0.05). However, the changes in MVR did not correlate with those in PRA, PAC, PNA or PTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Selenium deficiency as a cause of overload of iron and unbalanced distribution of other minerals.

Previous studies have shown that there are hematological abnormalities in selenium (Se)-deficient animals. This study examined the effects of Se deficiency on various minerals in serum and other tissues of male Wistar rats. The animals were given free access to either Torula yeast-based Se-deficient (SeD) diet or Se-adequate (SeA) (containing 0.1 mg Se/kg diet as sodium selenite) diet. Blood was sampled after 12 and 24 weeks, and the rats were killed after 24 weeks, for the analysis of minerals in serum, liver, kidney, heart, and spleen. Analyses showed that Se deficiency affected the concentrations of magnesium, calcium, iron, copper, and zinc in selected tissues and serum. During the entire feeding period, serum iron concentration was 40-58% greater in SeD rats compared with SeA rats. The transferrin saturation with iron was significantly greater in SeD rats than in SeA rats (57-60% versus 30-31%). Iron concentrations in the tissues ranged from 1.1 to 2.5 times higher in SeD rats than in SeA rats (p < 0.05). Similarly but to a lesser extent, the concentrations of zinc and magnesium were significantly greater in the serum of SeD rats compared with SeA rats, and the concentrations of calcium was significantly higher in kidney and spleen and of copper in liver, while the concentration of magnesium was significantly lower in liver and kidney. These results suggest that Se deficiency may cause a secondary overload of iron and unbalanced distribution of other minerals.

A sterol with an unusual side chain from Anoectochilus koshunensis.

A new sterol with a non-conventional side chain has been isolated from the whole plant of Anoectochilus koshunensis, together with four known sterols, a megastigmane glucoside and 2'-deoxyadenosine. The structure of the new sterol was elucidated as 26-methylstigmasta-5,22,25, (27)-trien-3 beta-ol based on chemical and detailed spectroscopic evidence.

Effects of Houttuyniae cordata and refinery final molasses on the development of offensive odor in porcine small intestine during storage.

Porcine small intestine evolved a specific offensive odor only 0.5 to 1 day after storage at 20 degrees C. We investigated the effects of Houttuyniae cordata (dokudami), refinery final molasses (RFM), green tea, and brown sugar on the evolution of methylmercaptan and ethanol, which were the main components of the volatiles which evolved from porcine small intestine in storage. Furthermore, we determined their antibacterial effect and deodorant activity against methylmercaptan, as possible factors in reducing the offensive odor. Addition of those materials reduced the offensive odor during storage. In particular, dokudami, green tea, and RFM markedly suppressed the evolution of methylmercaptan. RFM was most effective in suppressing the growth of bacteria. Dokudami had the highest deodorant activity, comparable to that of perilla leaves. However, the retardation of methylmercaptan evolution in situ cannot be simply explained by either of deodorant or antibacterial effect. It seems likely that the combined action of both effects affects the evolution of methylmercaptan in situ.

Characterization and localization of mouse hypothalamic growth hormone-releasing factor and effect of gold thioglucose-induced hypothalamic lesions.

Hypothalamic growth hormone-releasing factor (GRF) in higher mammals, including human GRF, is a 44 amino acid residue peptide and is highly homologous in structure. By contrast, mouse GRF (mGRF) recently deduced by cDNA cloning consists of only 42 residues and shows relatively low homology to the GRFs of higher mammals and the same rodent species, rat. To characterize and localize the predicted mature mGRF peptide in the hypothalamus, we have generated its antiserum and developed a homologous radioimmunoassay. Immunoreactive mGRF in the acid hypothalamic extract was eluted as a single peak at a position identical to that of synthetic peptide on both gel filtration chromatography and reverse-phase high-performance liquid chromatography (HPLC). Secretion of immunoreactive mGRF from incubated hypothalami increased several fold in response to 50 mM K+, and this rise was abolished in the absence of medium Ca2+. Only a single peak of immunoreactive mGRF that coeluted with synthetic replicate was observed after the K(+)-stimulated medium was extracted on Bond Elut C18 cartridges and applied on reverse-phase HPLC. Immunohistochemistry identified many mGRF-positive cell bodies in the arcuate nucleus and dense bundles of immunoreactive fibers in the median eminence. Treatment of mice with gold thioglucose (GTG), a chemical agent known to cause hypothalamic lesions, markedly depleted both content and in vitro secretion of immunoreactive mGRF. The decline in mGRF secretion was greater in GTG obese than in nonobese mice, whereas somatostatin secretion was not affected by GTG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

tert-butyl hydroperoxide-induced hemolysis of alpha-tocopherol-decreased erythrocytes from selenium-deficient and selenium-adequate rats.

The protective function of alpha-tocopherol, glutathione (GSH), and glutathione peroxidase (GSH-Px) from tert-butyl hydroperoxide (t-BuOOH)-induced hemolysis was studied with the erythrocytes from male Wistar rats fed selenium (Se)-adequate or -deficient diet for 3 months. By the preincubation with a water-soluble radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), at 10 mM for 6 h at 37 degrees C, alpha-tocopherol levels of the erythrocytes were decreased to 40% of the original level, that is, to the level insufficient for supporting the normal functions of the erythrocytes. With the Se-deficient cells, the hemolysis proceeded rapidly irrespective of the presence or absence of GSH in the incubation medium, and irrespective of the presence or absence of AAPH in the preincubation medium. Contrarily, GSH suppressed the hemolysis of Se-adequate cells which were preincubated with and without AAPH. These results are consistent with the notion that Se serves as the prime, important defense mechanism in the t-BuOOH-induced hemolysis through the activity of GSH-Px. Either alpha-tocopherol or GSH by itself, or both by themselves, may not play so significant a role as Se does in suppressing the hemolysis.

Deterioration of membrane morphology, phospholipids, and cytoskeletal protein in rat erythrocytes exposed to tert-butyl hydroperoxide: protection by exogenous glutathione fails in selenium deficiency.

Here we report scanning electron microscopy, phospholipid and fatty acid composition and cytoskeleton proteins of erythrocytes from Se-adequate and Se-deficient rats, showing protection by glutathione (GSH) against tert-butyl hydroperoxide (t-BuOOH)-induced hemolysis of erythrocytes from Se-adequate, but not from Se-deficient rats. Without exogenous GSH, erythrocytes incubated with t-BuOOH exhibited remarkable deterioration of cell membranes with diminished membrane phosphatidyl-ethanolamine, -serine, and -inositol and selective loss of a cytoskeletal protein, ankyrin 2-1. Without exogenous GSH these changes occurred in erythrocytes from both Se-deficient and Se-adequate rats. Dietary Se, through provision of glutathione peroxidase (GSH-Px) in erythrocytes as a probable scavenger of t-BuOOH, protects against hemolysis when GSH is available.

Variation of glutathione level and synthesis activity in chick liver due to selenium and vitamin E deficiencies.

Chicks were fed an amino acid mixture-based diet (basal diet) or one supplemented with selenium (Se, 0.2 micrograms/g as Na2SeO3) and/or vitamin E (100 micrograms/g as alpha-tocopherol). The group receiving the basal diet devoid of Se and vitamin E showed a tendency to grow slowly, but not significantly so, compared to the non-deficient control and manifested a symptom of exudative diathesis after the feeding period of 4 weeks. Supplementation of the basal diet with Se or vitamin E prevented the deficiency symptoms in the chicks. The hepatic GSH level and GSH synthesis activity were about three times as much in the Se- and vitamin E-deficient group as in the control. This was also the case for in vivo sulfur incorporation into hepatic GSH for 10 h post-injection with [35S]methionine. The increased level of GSH may partly compensate the hepatocytes for peroxidative damage.

Determination of nutritional efficiency of selenium contained in processed skipjack meat by comparison with selenite.

The nutritional efficiency of selenium (Se) contained in two kinds of processed fish meat was appraised. Rats were fed on a 20% casein diet deficient in Se (0.046 micrograms/g diet) for 2 weeks, and were then fed on the basal diet supplemented with 0.08 micrograms/g of Se as sodium selenite, boiled meat of skipjack or dried strip of skipjack for an additional 8 days. The Se-supplementation caused a significant increase of the Se concentration and the glutathione peroxidase activity in the rat liver. Although significant differences in hepatic Se levels were not observed among the rats fed on the Se-supplemented diets, the elevation of the hepatic enzyme activities of the rats fed on the skipjack-supplemented diets was only 45 to 53% that of the rats fed on the selenite-supplemented diet. Amounts of excretion of both fecal and urinary Se of the rats fed on the diets supplemented with the skipjacks were higher than those of the selenite-administered rats. These results indicate that the nutritional efficiency of the Se in the skipjack meat is about 50% that of selenite and that unknown factor(s) other than luminal absorption contribute to the low availability of the Se in the skipjack meat.