Effects of acivicin on growth, mycotoxin production and virulence of phytopathogenic fungi.

Acivicin is an inhibitor of γ-glutamyl transpeptidase and glutamine amidotransferase. When grown on a synthetic minimal agar medium, acivicin strongly inhibited the growth of Magnaporthe oryzae and Alternaria brassicicola, and to a lesser extent, Botrytis cinerea. However, only partial or marginal growth inhibition was observed with regard to Fusarium sporotrichioides and Fusarium graminearum. The growth retardation caused by acivicin was significantly alleviated by cultivating the fungus on a nutrient-rich medium. The inhibition of M. oryzae growth caused by 1 µmol l(-1) of acivicin on minimal agar medium was subdued by the addition of specific single amino acids, including His, a branched-chain amino acid (Leu, Ile or Val), an aromatic amino acid (Trp, Tyr or Phe), Met or Gln, at a concentration of 0·4 mmol l(-1). Trichothecene production by F. graminearum in trichothecene-inducing liquid medium was reduced significantly in the presence of acivicin despite its inability to inhibit growth in the trichothecene-inducing liquid medium. Foliar application of conidia in the presence of acivicin reduced the severity of rice blast disease caused by M. oryzae. These results suggest the usefulness of this modified amino acid natural product to mitigate agricultural problems caused by some phytopathogenic fungi. Significance and impact of the study: Fusarium head blight or scab disease and rice blast, caused by Fusarium graminearum and Magnaporthe oryzae, respectively, are major diseases of cereal crops that cause a significant loss of yield and deterioration in the quality of the grain. The present study investigated the effects of acivicin, a glutamine amino acid analog, on the physiology of various phytopathogenic fungi. Application of acivicin to a fungal culture and conidial suspension reduced mycotoxin production by the wheat scab fungus and the severity of rice blast, respectively. These results suggest the possibility that acivicin may serve as a lead compound to develop agricultural chemicals for the control of some plant diseases.

Hollow spheroids in ascites of ovarian clear cell carcinoma: how are they formed and how do they behave?

OBJECTIVE: Although the multicellular aggregates (spheroids) in malignant ascites are usually solid throughout, they sometimes have acellular hollow spaces, especially in ascites of ovarian clear cell carcinoma. The purpose of this study is to analyse the origin and behaviour of hollow spheroids. METHODS: Archival cytological and histological specimens of 32 ovarian carcinomas, including 12 clear cell carcinomas, were reviewed. HAC-2, a clear cell carcinoma cell line, was injected into the abdominal cavity of nude mice for direct comparison of ascitic cytology and tumour histology. Spheroids that were collected from nude mice ascites were cultured in vitro to observe their behaviour. RESULTS: Five of six clear cell carcinomas with hollow spheroids showed spherule-like hyaluronan-rich stroma in their tumour tissue, whereas those without hollow spheroids did not. After heterotransplantation, both ascites and tumour imprints showed small or large hollow spheroids. Hyaluronan was detected in the former but not in the latter. The abdominal tumours showed compact spherule-like hyaluronan-rich stroma, enlarged oedematous stroma or intermediate stroma. In both size and hyaluronan status, small and large hollow spheroids were approximately comparable to spherule-like hyaluronan-rich stroma and oedematous stroma, respectively. During culture in vitro, hollow spheroids were maintained as hollow spheroids in suspension, and produced daughter hollow spheroids. CONCLUSIONS: The hollow space in the spheroids originates from spherule-like hyaluronan-rich stroma, where water trapping by hyaluronan causes enlargement of the space. The matrix within the hollow space serves as a scaffold that regulates cell polarity and matrix production.

Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3beta positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-kappaB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3beta and to assess the anti-cancer effect of GSK-3beta inhibition in RCC. METHODS: Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3beta in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT-PCR, BrDU incorporation and MTS assays to study the effect of GSK-3beta inactivation on renal cancer cell proliferation and survival. RESULTS: We detected aberrant nuclear accumulation of GSK-3beta in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-kappaB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells. CONCLUSIONS: Our results show nuclear accumulation of GSK-3beta as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.

Overexpression of osteopontin in clear cell carcinoma of the ovary: close association with HNF-1beta expression.

AIMS: Transcription factor hepatocyte nuclear factor (HNF)-1beta is selectively expressed in clear cell carcinoma (CCC) of the ovary. One of the potential HNF-1beta target genes is osteopontin (OPN). Although elevation of OPN mRNA has been reported in CCC, it remains unclear whether CCC cells overexpress OPN protein. The aim was to investigate the expression of OPN protein and its correlation with HNF-1beta status in CCC. METHODS AND RESULTS: Three CCC and two serous adenocarcinoma (SA) cell lines were evaluated for expression of OPN by reverse transcriptase-polymerase chain reaction and immunocytochemistry. OPN expression, at both the mRNA and protein levels, was higher in the three CCCs than in the two SAs. HNF-1beta expression was detected in the CCCs but not in the SAs. Subsequently, 60 surgical specimens (30 CCCs and 30 SAs) were examined immunohistochemically for expression of OPN and HNF-1beta. All 30 CCCs showed immunopositivity for both OPN and HNF-1beta. The 12 (40%) CCCs with a high OPN score all had a high HNF-1beta score. In contrast, SAs rarely showed immunoreactivity for OPN or HNF-1beta. CONCLUSIONS: OPN expression is elevated in ovarian CCC and is closely associated with HNF-1beta overexpression. HNF-1beta is likely to participate in OPN up-regulation in CCC.

E-Cadherin gene promoter hypermethylation in primary human gastric carcinomas.

BACKGROUND: E (epithelial)-cadherin, the cell adhesion molecule also considered a potential invasion/metastasis suppressor, is mutationally inactivated in nearly half of all undifferentiated-scattered (diffuse-type) gastric carcinomas. In addition, silencing of E-cadherin by CpG methylation within its promoter region has been reported in several gastric carcinoma cell lines. We investigated the methylation status of the E-cadherin promoter region in 53 primary human gastric carcinomas. METHODS: Hypermethylation of the E-cadherin promoter was determined by utilizing methylation-specific polymerase chain reaction (PCR)-single-strand conformation polymorphism (MSP-SSCP) analysis followed by direct sequencing of PCR products. Expression of E-cadherin was studied by western blot analysis. All statistical tests were two-sided. RESULTS: Hypermethylation of the E-cadherin promoter was evident in 27 (51%) of 53 primary gastric carcinomas examined by MSP-SSCP. It occurred more frequently in carcinomas of the undifferentiated-scattered type (in 15 [83%] of 18) than in other histologic subtypes (in 12 [34%] of 35) (P =.0011, Fisher's exact test), and it was present at similar rates in early (in six [60%] of 10) versus advanced (in 21 [49%] of 43) carcinomas (P =.73, Fisher's exact test). Methylation occurring at all cytosine-guanosine sequences (CpGs) near the transcriptional start site was confirmed in six of six tumors examined by bisulfite-DNA sequencing, including two early gastric carcinomas. In addition, loss or diminished expression of E-cadherin was confirmed by western blotting in four of the six tumor tissues demonstrating hypermethylation. CONCLUSIONS: The E-cadherin promoter frequently undergoes hypermethylation in human gastric cancers, particularly those of the undifferentiated-scattered histologic subtype. E-cadherin promoter hypermethylation is associated with decreased expression and may occur early in gastric carcinogenesis.

Biosynthesis of an interstitial type of collagen by cloned human gastric carcinoma cells.

In an attempt to elucidate the role played by neoplastic epithelial cells in the formation of stromal collagen, the synthesis of collagen by two cloned human gastric carcinoma cell lines was studied. The presence of antigenicity of procollagen alpha 1(I) chain in the cytoplasms of both carcinoma cell lines growing in culture was demonstrated by an immunocytochemical technique using specific antibodies. After denaturation of the radiolabeled collagenous proteins extracted from the combined cells and culture media, two components comigrating with authentic alpha 1(I) and alpha 2 chains on sodium dodecyl sulfate:polyacrylamide gel electrophoresis were found. Electrophoretogram analysis revealed further a dense band slightly slower than the alpha 1(I) chain, most likely representing alpha 1(I) trimer. These radioactive components disappeared after exposure of the samples to bacterial collagenase. The relative activity of collagen synthesis determined by using purified collagenase was slightly higher than that of fibroblasts derived from human synovial membrane in culture. The same antibodies to procollagen alpha 1(I) chain also labeled the cytoplasms of carcinoma cells, and extracellular matrix of the tumors developed after transplantation of one of the cell lines into the nude mice. Our data indicate that both human gastric carcinoma cell lines synthesize type 1 collagen in vivo as well as in vitro and suggest that carcinoma cells may play an active role in the formation of stromal collagen in most human carcinomas.

Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia.

The adenomatous polyposis coli (APC) tumor suppressor gene is mutationally inactivated in both familial and sporadic forms of colorectal cancers. In addition, hypermethylation of CpG islands in the upstream portion of APC, a potential alternative mechanism of tumor suppressor gene inactivation, has been described in colorectal cancer. Because a subset of both gastric and colorectal cancers display the CpG island methylator phenotype, we hypothesized that epigenetic inactivation of APC was likely to occur in at least some gastric cancers. APC exhibits two forms of transcripts from exons 1A and 1B in the stomach. Therefore, we investigated CpG island methylation in the sequences upstream of exons 1A and 1B, i.e., promoters 1A and 1B, respectively. We evaluated DNAs from 10 gastric cancer cell lines, 40 primary gastric cancers, and 40 matching non-cancerous gastric mucosae. Methylated alleles of promoter 1A were present in 10 (100%) of 10 gastric cancer cell lines, 33 (82.5%) of 40 primary gastric cancers, and 39 (97.5%) of 40 noncancerous gastric mucosae. In contrast, promoter 1B was unmethylated in all of these same samples. APC transcripts from exon 1A were not expressed in nine of the 10 methylated gastric cancer cell lines, whereas APC transcripts were expressed from exon 1B. Thus, expression from a given promoter correlated well with its methylation status. We conclude that in contrast to the colon, methylation of promoter 1A is a normal event in the stomach; moreover, promoter 1B is protected from methylation in the stomach and thus probably does not participate in this form of epigenetic APC inactivation.

T-786-->C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm.

BACKGROUND: Coronary spasm plays an important role in the pathogenesis of ischemic heart diseases in general. However, the precise mechanism(s) responsible for coronary spasm remains to be elucidated, and we examined the molecular genetics of coronary spasm. METHODS AND RESULTS: We searched for the possible mutations in the endothelial nitric oxide synthase (eNOS) gene in patients with coronary spasm. In this study, we demonstrate the existence of 3 linked mutations in the 5'-flanking region of the eNOS gene (T-786-->C, A-922-->G, and T-1468-->A). The incidence of the mutations was significantly greater in patients with coronary spasm than in the control group (P<0.0001). Multiple logistic regression analysis with forward stepwise selection using the environmental risk factors and the eNOS gene variant revealed that the most predictive independent risk factor for coronary spasm was the mutant allele (P<0.0001). As assessed by luciferase reporter gene assays, the T-786-->C mutation resulted in a significant reduction in eNOS gene promoter activity (P<0.05), whereas neither the A-922-->G nor the T-1468-->A mutation had any affect. CONCLUSIONS: Taken together, these findings strongly suggest that the T-786-->C mutation in the eNOS gene reduces the endothelial NO synthesis and predisposes the patients with the mutation to coronary spasm.

Estradiol supplementation suppresses hyperventilation-induced attacks in postmenopausal women with variant angina.

OBJECTIVES: We sought to examine whether estradiol (E2) supplementation suppresses anginal attacks in women with variant angina. BACKGROUND: Estrogen is known to improve endothelial function. Coronary spasm plays an important role in the pathogenesis of not only variant angina but also ischemic heart disease in general, and endothelial dysfunction seems to be involved in the pathogenesis of coronary spasm. METHODS: Fifteen postmenopausal women with variant angina (mean age 54.2 years) were given a hyperventilation (HV) test, a provocation test for coronary spasm, in the early morning of day 1 (baseline), day 3 (after 2-day transdermal E2 supplementation, 4 mg) and day 5 (after 2-day placebo administration). We measured the flow-mediated (endothelium-dependent) dilation (FMD) of the brachial artery with the ultrasound technique before each HV test. RESULTS: The anginal attacks with ST segment elevation were induced by HV in all patients on days 1 and 5. However, no attacks were induced on day 3. Supplementation with E2 augmented FMD (3.5 +/- 0.6*, 8.9 +/- 0.7 and 4.0 +/- 0.5* on days 1, 3 and 5, respectively; *p < 0.01 vs. day 3). The serum E2 levels on days 1, 3 and 5 were 22.7 +/- 2.8*, 96.2 +/- 9.2 and 30.7 +/- 7.1* pg/ml, respectively (*p < 0.01 vs. day 3). CONCLUSIONS: The present results demonstrated for the first time, to our knowledge, that E2 supplementation suppresses the HV-induced attacks in women with variant angina, in part because of the improvement of endothelial function.

Improvement of endothelial function and insulin sensitivity with vitamin C in patients with coronary spastic angina: possible role of reactive oxygen species.

OBJECTIVES: This study was designed to examine the effect of antioxidant supplementation on the endothelial function and insulin sensitivity in patients with coronary spastic angina (CSA). BACKGROUND: Insulin resistance may play a key role in coronary heart disease, and there is a possible link between acetylcholine-induced coronary vasoconstriction and hyperinsulinemia in patients with CSA. Endothelial dysfunction is present in the systemic arteries in CSA patients, and reactive oxygen species may cause inactivation of nitric oxide in these patients. METHODS: We measured flow-mediated dilation of the brachial artery using ultrasound technique in 22 patients with CSA and 20 control subjects. We also evaluated glucose tolerance using a 75-g oral glucose tolerance test and insulin sensitivity using steady-state plasma glucose (SSPG) methods in the same patients. RESULTS: The incidence of impaired glucose tolerance was higher in the CSA group than in the control group. Vitamin C infusion augmented flow-mediated dilation and decreased SSPG levels in the CSA group (from 3.27 +/- 0.77% to 7.00 +/- 0.59% [p < 0.001 by analysis of variance (ANOVA)] and from 177.3 +/- 13.3 to 143.1 +/- 14.9 mg/dl [p = 0.047 by ANOVA], respectively) but not in the control group (from 6.47 +/- 0.66% to 6.80 +/- 0.60% and from 119.8 +/- 11.7 mg/dl to 118.1 +/- 11.3 mg/dl, respectively). The steady-state plasma insulin levels were not affected by vitamin C infusion in either group. CONCLUSIONS: Vitamin C improves both endothelial function and insulin sensitivity in patients with CSA. Thus, reactive oxygen species and/or decreased nitric oxide bioactivity may play an important role in the genesis of both endothelial dysfunction and insulin resistance in patients with CSA.

Deficiency in nitric oxide bioactivity in epicardial coronary arteries of cigarette smokers.

OBJECTIVES: This study sought to examine nitric oxide-mediated regulation of epicardial coronary arterial tone in cigarette smokers. BACKGROUND: Cigarette smoking is a major risk factor for coronary artery disease and is highly prevalent in patients with coronary spastic angina. Long-term exposure to cigarette smoking has been recently reported to suppress endothelium-dependent arterial relaxation in vivo humans. METHODS: Responses of epicardial coronary artery diameter to single or combined infusion of acetylcholine and NG-monomethyl-L-arginine (L-NMMA) into the left main coronary artery were examined in 11 current smokers and 17 nonsmokers using quantitative coronary angiography. RESULTS: Acetylcholine dilated one-third of the proximal segments and most of the distal segments of coronary arteries in nonsmokers, whereas it constricted most of the proximal and distal segments in smokers. L-NMMA decreased the basal diameter of coronary arteries in nonsmokers but had minimal effect on the basal diameter in smokers. L-NMMA abolished the dilator response to acetylcholine in the coronary arteries of nonsmokers but had minimal effect on the constrictor response to acetylcholine in the arteries of smokers. The dilator response to nitroglycerin was significantly increased in the coronary arteries of smokers compared with in those of nonsmokers. The constrictor response to L-NMMA at rest was significantly correlated with the dilator response to nitroglycerin and with the diameter changes to acetylcholine in both smokers and nonsmokers. CONCLUSIONS: Nitric oxide bioactivity at rest and at acetylcholine-stimulated conditions in smokers was decreased, leading to the supersensitivity of the artery to the dilator effect of nitroglycerin as well as the constrictor effect of acetylcholine in smokers. Cigarette smoking affects nitric oxide-mediated regulation of coronary artery tone.

Gender difference in improvement of endothelium-dependent vasodilation after estrogen supplementation.

OBJECTIVES: We examined whether there is a gender difference in the improvement of endothelium-dependent vasodilation after estrogen supplementation. BACKGROUND: Estrogen therapy reduces the risk of cardiovascular events in postmenopausal women, and the augmented release of endothelium-derived nitric oxide (NO) by estrogens has been suggested to be one of the mechanisms for the cardioprotective effects of estrogen. METHODS: With ultrasound technique, we measured the diameter and blood flow of the brachial artery at rest, during reactive hyperemia after transient occlusion and after nitroglycerin administration before and after estradiol supplementation in 15 postmenopausal women (mean 63 years) and in 15 men matched for age and risk factors for atherosclerosis. RESULTS: Estradiol supplementation augmented the flow-mediated vasodilation and serum level of nitrite/nitrate (metabolites of NO) in women (respectively, from mean +/- SEM of 8.0 +/- 0.6% to 12.9 +/- 0.6% [p < 0.01 by analysis of variance (ANOVA)] and from 64.9 +/- 8.7 to 93.7 +/- 9.4 mumol/liter [p < 0.05 by ANOVA]) but not in men (respectively, from 8.1 +/- 0.6% to 8.3 +/- 0.7% and from 57.8 +/- 6.7 to 60.8 +/- 5.4 mumol/liter). The increases in blood flow during reactive hyperemia and in diameter after nitroglycerin administration were not affected by estradiol supplementation in either men or women. CONCLUSIONS: Estradiol supplementation improves endothelium-dependent vasodilation in women, probably because of augmented NO production/release, but not in men. Thus, there may be gender differences in the effects of estrogen therapy on endothelial functions and NO production/release.

Diffuse intimal thickening of coronary arteries in patients with coronary spastic angina.

OBJECTIVES: The purpose of this study was to evaluate the extent of atherosclerotic changes in angiographically normal coronary arteries using intravascular ultrasound (IVUS) technique in patients with coronary spastic angina. BACKGROUND: Nitric oxide activity was shown to be decreased in coronary arteries of patients with coronary spastic angina (CSA). Decrease in nitric oxide causes arterial intimal hyperplasia or thickening. However, it remains unclear whether intimal thickening is diffusely present in coronary arteries of patients with CSA. METHODS: The IVUS study was performed in 26 patients with CSA and with normal coronary angiograms and in 31 control subjects in whom age and gender was matched with those in patients with CSA. RESULTS: Compared with control subjects, patients with CSA had significantly larger percent intima + media area (%I + M area), intima + media area and maximal intima + media thickness in all of proximal, middle and distal segments (p<0.01, respectively). Lumen area was comparable between these groups. The presence of spasm was the most powerful independent predictor of increase in percent intima + media area, in multiple-regression analysis with the traditional risk factors as covariates. CONCLUSIONS: Intimal thickening existed entirely in a coronary artery in patients with CSA and with normal angiograms, independently of other traditional risk factors. The diffuse intimal thickening in the spasm coronary arteries is intimately related with coronary spasm.

Vitamin E administration improves impairment of endothelium-dependent vasodilation in patients with coronary spastic angina.

OBJECTIVES: We examined the effects of oral administration of vitamin E, an antioxidant, on endothelium-dependent vasodilation in patients with coronary spastic angina. BACKGROUND: We have recently reported that endothelium-dependent vasodilation is impaired in patients with coronary spastic angina (CSA). Furthermore, it is known that oxidative stress may play an important role in the impairment of endothelium-dependent vasodilation in cardiovascular diseases. METHODS: With the ultrasound technique, flow-dependent vasodilation of the brachial arteries during reactive hyperemia was examined before and after treatment for a month with either oral administration of vitamin E (alpha-tocopherol acetate, 300 mg/day) or placebo, which is randomly assigned, in patients with CSA (n=60). RESULTS: Before treatment, patients with CSA had impaired flow-dependent vasodilation, lower plasma levels of alpha-tocopherol and higher plasma levels of thiobarbituric acid reactive substances (TBARS), as compared with age- and sex-matched control subjects (n=60) (flow-dependent vasodilation: 3.1+/-1.8 vs. 7.1+/-2.5%, p < 0.001; alpha-tocopherol levels: 8.9+/-1.8 vs. 10.8+/-1.8 microg/ml, p < 0.001). In patients with CSA, treatment with vitamin E restored flow-dependent vasodilation (3.1+/-1.7 vs. 8.3+/-2.0%, p < 0.001), and this improvement was associated with the decreases in plasma TBARS levels and anginal attacks. CONCLUSIONS: The results indicate that vitamin E treatment improved endothelium-dependent vasodilation and decreased plasma TBARS levels in patients with CSA. Thus, increased oxidative stress may contribute to endothelial dysfunction and anginal attacks in patients with CSA.

Vitamin C attenuates abnormal vasomotor reactivity in spasm coronary arteries in patients with coronary spastic angina.

OBJECTIVES: This study sought to examine effect of vitamin C, an antioxidant, on the abnormal vasomotor reactivity in spasm coronary arteries. BACKGROUND: Oxygen free radicals generated in the arterial walls have been shown to cause endothelial vasomotor dysfunction. METHODS: Responses of the epicardial arterial diameters of the left coronary arteries to the intracoronary infusion of acetylcholine (ACh) (10 and 50 microg/min) were measured by quantitative coronary angiography before and during combined intracoronary infusion of vitamin C (10 mg/min) or saline as a placebo in 32 patients with coronary spastic angina and in 34 control subjects. RESULTS: Vitamin C infusion suppressed the constrictor response of the epicardial diameter to ACh in spasm coronary arteries but had no significant effect in the control coronary arteries (percent change in distal diameter in response to 10 microg/min of ACh [constriction (-), dilation (+), mean +/- SEM] before vitamin C: -8.2 +/- 2.9% in spasm arteries, +8.4 +/- 2.9%* in control arteries; during vitamin C: +0.2 +/- 3.8%* in spasm arteries, +7.2 +/- 1.3%* in control arteries [*p < 0.01 vs. spasm arteries before vitamin CI). The coronary sinus-arterial difference in plasma thiobarbituric acid reactive substances during ACh infusion, an indicator of lipid peroxidation in coronary circulation, was higher in patients with coronary spastic angina than in control subjects (p < 0.01) but was suppressed in patients with coronary spastic angina to comparable levels in control subjects by combined infusion of vitamin C. Saline infusion had no effect. CONCLUSIONS: The results indicate that vitamin C attenuates vasomotor dysfunction in epicardial coronary arteries in patients with coronary spastic angina. Oxygen free radicals may at least in part play a role in the abnormal coronary vasomotor reactivity in response to ACh in spasm coronary arteries.

Hyperglycemia rapidly suppresses flow-mediated endothelium-dependent vasodilation of brachial artery.

OBJECTIVES: We examined whether endothelial dysfunction occurs when acute hyperglycemia is induced by oral glucose loading. BACKGROUND: Endothelial dysfunction has been shown to occur in patients with diabetes mellitus (DM), and chronic hyperglycemia is implicated as a cause of endothelial dysfunction. However, in many patients with Type 2 DM and in those with impaired glucose tolerance (IGT), fasting blood glucose may be within normal limits, and hyperglycemia occurred only post-prandially. METHODS: With ultrasound technique, we measured flow-mediated endothelium-dependent vasodilation during oral glucose tolerance test in 58 subjects: (17 patients with normal glucose tolerance [NGT], 24 with IGT, and 17 with type 2 DM). In addition, we measured the levels of thiobarbituric acid reactive substances (TBARS) and nitrite/nitrate. RESULTS: Flow-mediated vasodilation decreased after glucose loading (NGT: 7.53+/-0.40, 4.24+/-0.28 and 6.35+/-0.40, in fasting, at 1- and 2-h, respectively, IGT: 6.50+/-0.48, 1.40+/-0.41** and 4.00+/-0.47*, respectively; DM: 4.77+/-0.37, 1.35+/-0.38** and 1.29+/-0.29%**, respectively; *p < 0.01 vs. fasting, **p < 0.005 vs. fasting). The TBARS concentration increased in parallel with plasma glucose level in each group (NGT: 1.43+/-0.07, 2.03+/-0.12 and 1.80+/-0.12, respectively; IGT: 1.65+/-0.11, 2.46+/-0.12** and 1.94+/-0.08*, respectively; DM: 1.73+/-0.07, 2.34+/-0.08** and 2.47+/-0.09** nmol/ml, respectively; *p < 0.05 vs. fasting, **p < 0.01 vs. fasting). Glucose loading did not change nitrite/nitrate concentration in any of the groups. CONCLUSIONS: Hyperglycemia in response to oral glucose loading rapidly suppresses endothelium-dependent vasodilation, probably through increased production of oxygen-derived free radicals. These findings strongly suggest that prolonged and repeated post-prandial hyperglycemia may play an important role in the development and progression of atherosclerosis.

Nitric oxide-mediated flow-dependent dilation is impaired in coronary arteries in patients with coronary spastic angina.

OBJECTIVES: This study sought to examine whether flow-dependent dilation is impaired at the site of coronary artery spasm in patients with coronary spastic angina. BACKGROUND: Physiologic stimuli such as exercise and exposure to cold have been shown to cause an increase in coronary blood flow, leading to flow-dependent dilation of coronary arteries in normal subjects, but cause coronary constriction in patients with coronary spastic angina. METHODS: A maximal increase in blood flow was induced selectively in the left anterior descending coronary artery (LAD) by infusion of adenosine through a Doppler flow catheter tip in the midportion of the LAD in 10 patients with coronary spastic angina, all with angiographically demonstrated spasm of the LAD, and in 11 control patients. Coronary artery diameter at the proximal site of the LAD (exposed to increased flow but not to adenosine) was measured by quantitative angiography. RESULTS: Flow-dependent dilation of the proximal LAD was found to be less in spasm arteries than in control arteries. Infusion of NG-monomethyl-L-arginine (L-NMMA) in the proximal LAD suppressed flow-dependent dilation in control arteries but had no significant effect on spasm arteries. The dilator response to nitroglycerin was not impaired in spasm coronary arteries. CONCLUSIONS: Our results indicate that flow-dependent coronary dilation is impaired in spasm arteries, partly due to a deficiency in endothelial nitric oxide bioactivity, which in turn may contribute to the increase in coronary tone during physiologic stimuli in patients with coronary spastic angina.

Improvement of endothelial vasomotor dysfunction by treatment with alpha-tocopherol in patients with high remnant lipoproteins levels.

OBJECTIVES: This study sought to examine whether oral intake of alpha-tocopherol, an antioxidant, could improve endothelium-dependent vasorelaxation in patients with high remnant lipoproteins levels. BACKGROUND: Remnant lipoproteins are known to be atherogenic and impair endothelium-dependent arterial relaxation, but the underlying mechanisms remain unclear. Oxidative stress is a common feature of various risk factors for atherosclerosis. METHODS: Flow-mediated vasodilation of the brachial artery during reactive hyperemia was examined by high resolution ultrasound technique before and at the end of 4 weeks treatment with oral administration of alpha-tocopherol acetate (300 IU/day) or placebo, which was randomly assigned, in 40 patients with high serum levels of remnants and in 30 patients with low remnants levels in the fasting state (>75th percentile and <25th percentile, respectively, of the distribution of remnants levels in 150 consecutive hospitalized patients). RESULTS: Before treatment, flow-mediated vasodilation was lower in patients with high remnants levels than in those with low levels (4.1 +/- 0.3% vs. 6.0 +/- 0.5%, p < 0.01). Treatment with alpha-tocopherol but not with placebo significantly increased flow-mediated dilation in patients with high remnants levels (7.5 +/- 0.4% after alpha-tocopherol vs. 4.2 +/- 0.4% after placebo, p < 0.01). In patients with low remnants levels, alpha-tocopherol was not effective. The beneficial effect with alpha-tocopherol in high remnants patients was associated with decrease in plasma levels of thiobarbituric acid reactive substances, an indicator of lipid peroxidation (6.6 +/- 0.3 nmol/ml before alpha-tocopherol vs. 4.6 +/- 0.3 after alpha-tocopherol, p < 0.05). CONCLUSIONS: Alpha-tocopherol improved impairment of endothelium-dependent vasodilation in patients with high remnants levels. The increase in oxidative stress may at least partly contribute to endothelial vasomotor dysfunction, in patients with high remnants levels.

Oral magnesium supplementation in patients with essential hypertension.

To elucidate the effects of magnesium on high blood pressure, a 4-week study of oral magnesium supplementation (MgO 1 g/day) was conducted in 21 outpatients with uncomplicated essential hypertension. During the study, blood pressure and intraerythrocyte sodium concentration decreased significantly, and the erythrocyte ouabain-sensitive 22Na efflux rate constant (Kos) and intraerythrocyte magnesium concentration both increased. Serum triglyceride and free fatty acid concentrations were reduced. Furthermore, the elevation in Kos significantly and positively correlated with both the increase in intraerythrocyte magnesium concentration and the decrease in mean blood pressure. There was a significant inverse correlation between the prestudy Kos and the decrease in mean blood pressure. In addition, when patients were divided according to their overall decrease in mean blood pressure, the prestudy intraerythrocyte sodium concentration was significantly higher in patients with a mean blood pressure decrease of more than 7 mm Hg than that of patients whose mean blood pressure decrease was less than 7 mm Hg. These results suggest that oral magnesium supplementation may lower blood pressure through the activation of a cell membrane sodium pump and may reduce serum lipid concentration. It also suggests that the lower the prestudy Kos or the higher the prestudy intraerythrocyte sodium concentration, the more effective the oral magnesium treatment is in lowering blood pressure. Therefore, we concluded that appropriate oral magnesium intake might be effective as a nonpharmacological treatment for essential hypertension.

Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.

The non-coding control region of mitochondrial DNA (mtDNA), containing the hypervariable regions HV1 and HV2 and the D-loop region, was screened for mutations in 45 gastric tumours (15 tumours each of adenoma, differentiated adenocarcinoma and undifferentiated carcinoma). We found mutations in two of the 45 tumours (4%); a 1 bp A deletion at nucleotide position 248 in a differentiated adenocarcinoma and a G to A transition at nucleotide position 16,129 in an adenoma. We also observed 10 polymorphisms, four of which were not previously recorded. Both mtDNA mutations were present in replication error negative (RER-) tumours. Short mono- or dinucleotide repeats in the control region, such as (C)7, (A)5 or (CA)5, were not altered regardless of nuclear genetic instability. In summary, mtDNA is mutated in a subset of benign and malignant gastric tumours, but, disruption of the mtDNA repair system appears not to be significantly involved in gastric tumours of Japanese patients.