The red wine phenolics piceatannol and myricetin act as agonists for estrogen receptor alpha in human breast cancer cells.

Previous epidemiological reports have suggested that red wine intake is associated with beneficial health effects due to the ability of certain phytochemical components to exert estrogen-like activity. It has been also documented that estrogens induce the proliferation of hormone-dependent breast cancer cells by binding to and transactivating estrogen receptor (ER) alpha, which in turn interacts with responsive DNA sequences located within the promoter region of target genes. In order to provide further insight into the positive association between wine consumption and the incidence of breast carcinoma in postmenopausal women, we have evaluated the estrogenic properties of two abundant wine-derived compounds, named piceatannol (PIC) and myricetin (MYR), using as model systems the hormone-sensitive MCF7 and the endocrine-independent SKBR3 breast cancer cells. On the basis of our experimental evidence PIC and MYR may contribute to the estrogenicity of red wine since: (1) they transactivate endogenous ER alpha; (2) they activate the agonist-dependent activation function (AF) 2 of ER alpha and ER beta in the context of the Gal4 chimeric proteins; (3) they rapidly induce the nuclear immunodetection of ER alpha; (4) they regulate the expression of diverse estrogen target genes; (5) they compete with 17beta-estradiol for binding to ER alpha and ER beta; and--as a biological counterpart of the aforementioned abilities--(6) they exert stimulatory effects on the proliferation of MCF7 cells. Hence, the estrogenic activity of PIC and MYR might be considered at least as a potential factor in the association of red wine intake and breast tumors, particularly in postmenopausal women.

Isolation of a MADS-box gene (ERAF17) and correlation of its expression with the induction of formation of female flowers by ethylene in cucumber plants (Cucumis sativus L.).

Ethylene regulates sex expression in cucumber (Cucumis sativus L.) plants. When the apices of monoecious cucumber seedlings (cv. Shimoshirazu-jibai) were treated with the ethylene-releasing compound, ethephon, female flowers were induced at the nodes. To clarify the action of ethylene in the regulation of sex expression, we attempted to isolate genes whose expression changed during induction of the formation of female flowers at the apices of these cucumber plants upon treatment with ethephon. Using the differential-display method, we identified 20 clones (#1 to #20) that reflected differences in the accumulation of transcripts in apices treated or not treated with ethephon. Sequence analysis of cDNA fragments revealed that the cDNA #17 had the sequence of a MADS-box gene. We isolated the full-length cDNA and showed that it included both a MADS box and a K box, and the corresponding gene was designated ERAF17. We examined the expression of ERAF17 in the apices of cv. Shimoshirazu-jibai and in those of a gynoecious cultivar (Rensei). In these cultivars, the timing and levels of expression of the ERAF17 transcript were correlated with the development of female flowers. Induction of the synthesis of the ERAF17 transcript by ethephon occurred within 4 h of the start of treatment and continued for 4 days at least. Expression of ERAF17 at apices was localized in the floral buds of the gynoecious cultivar, and expression was maintained in female flowers thorough their development. Our results suggest that the induction of the formation of female flowers by ethylene might be regulated by the expression of ERAF17 in floral buds at the apices of cucumber plants and that expression of this gene might also be involved in the development of female flowers.

Enhancement of learning capacity and cholinergic synaptic function by carnitine in aging rats.

The effects of a carnitine derivative, acetyl-L-carnitine (ALCAR), on the cognitive and cholinergic activities of aging rats were examined. Rats were given ALCAR (100 mg/kg) per os for 3 months and were subjected to the Hebb-Williams tasks and a new maze task, AKON-1, to assess their learning capacity. The learning capacity of the ALCAR-treated group was superior to that of the control. Cholinergic activities were determined with synaptosomes isolated from the cortices. The high-affinity choline uptake by synaptosomes, acetylcholine synthesis in synaptosomes, and acetylcholine release from synaptosomes on membrane depolarization were all enhanced in the ALCAR group. This study indicates that chronic administration of ALCAR increases cholinergic synaptic transmission and consequently enhances learning capacity as a cognitive function in aging rats.

Estrogen receptor alpha mediates the proliferative but not the cytotoxic dose-dependent effects of two major phytoestrogens on human breast cancer cells.

Phytoestrogens are a chemically diverse group of compounds made by plants that can have estrogenic effects in animals. Both tumorigenic and antitumorigenic effects have been reported. Although estrogens stimulate the growth of many breast tumors, there is a negative correlation between the incidence of breast cancer and the phytoestrogen-rich diet of certain Asian populations. To begin to resolve this paradox, we have analyzed the estrogenic properties of genistein and quercetin, two flavonoid phytoestrogens particularly abundant in soybeans. Trans-activation experiments with a transfected reporter gene for nuclear estrogen receptors (ER) show strong activation of the endogenous ER alpha by both phytoestrogens in two MCF7 human breast cancer cell lines. This is supported by the observation that the two phytoestrogens induce the down-regulation of ER alpha mRNA and protein levels. Using chimeric proteins consisting of the hormone binding domains of ER alpha and ER beta fused to the Gal4 DNA binding domain, we have established that genistein and quercetin are full estrogenic agonists of both ER isoforms. Ligand binding experiments with purified ER alpha and ER beta confirm that the two phytoestrogens are ER ligands. At concentrations that are sufficient to obtain substantial transcriptional activity, they stimulate the proliferation of two ER alpha-dependent breast cancer cell lines. At high concentrations, such as those reached with a soy-rich diet, genistein and quercetin are strong cytotoxic agents that even kill ER-independent HeLa cells. Thus, the mode of action of phytoestrogens and the balance between being risk or chemopreventive factors for breast cancer may depend on the dietary load.

Closed-loop control using a stretch sensor for restoration of standing with functional electrical stimulation in complete paraplegia.

A closed-loop control system for standing with functional electrical stimulation (FES) using percutaneous intramuscular electrodes in complete paraplegia is described. The system consisted of ultrafine percutaneous intramuscular electrodes, a 32-channel stimulator and a stretch sensor with active current control to detect knee buckling. The closed-loop control system was applied in a T8 completely paraplegic patient. Compared to the stretch sensor with a wide use flexible goniometer for direct current control during standing, the stretch sensor was superior to the flexible goniometer in both ease of use and response. The average time delay from the start of knee buckling until the sensor turned on was 0.56+/-0.19 seconds (Mean+/-S.D.) in the goniometer and 0.21+/-0.06 seconds in the stretch sensor. The average time delay from the start of knee buckling until the recovery from knee buckling was 1.01+/-0.05 seconds in the goniometer and 0.78+/-0.06 seconds in the stretch sensor. The continuous standing ability of the patient increased from 12 minutes with open-loop stimulation to 30 minutes with the closed-loop control. No complications such as falling occurred during clinical use. This system prevented falling due to knee buckling during standing and prolonged upright activities in complete paraplegics.

Effect of the long-term feeding of dietary lipids on the learning ability, fatty acid composition of brain stem phospholipids and synaptic membrane fluidity in adult mice: a comparison of sardine oil diet with palm oil diet.

The effect of 12 month feeding of 5% palm oil or sardine oil diet on the maze-learning ability, fatty acid composition of brain stem phospholipids and synaptic membrane fluidity in mice was studied. The time required to reach the maze exit and the number of times that a mouse strayed into blind alleys in the maze were measured three times every 4 days. The time and number of mice fed on the sardine oil diet were less than those of animals fed on the palm oil diet in the first and second trials. The results of fatty acid composition analysis of brain stem phosphatidylethanolamine showed that the percentage of docosahexaenoic acid (22:6, n-3; DHA) was higher, but the arachidonic acid (20:4, n-6; AA) and docosatetraenoic acid (22:4, n-6; DTA) were lower in the sardine oil diet fed-mice than in the palm oil diet fed-animals. Moreover, the microviscosity of the synaptic plasma membrane in the sardine oil diet group was lower than that in the palm oil diet group. These results suggest that the adult mice fed on the sardine oil diet for a long period maintain higher levels of docosahe xaenoic acid in brain phospholipids, synaptic membrane fluidity and maze-learning ability than animals fed on the palm oil diet.

Mineral concentrations in the blood of male smokers and non-smoking males and females measured with fluoro-X-ray analyzer.

For clarification of the effects of smoking on mineral absorption in red blood cells (RBC) and serum, their concentrations at these sites were examined in ten male smokers and 12 male and 10 female non-smokers by fluoro-X-ray analysis. K in serum was high, and P, Ca and Fe low, compared to RBC. Mg and S in serum and RBC were essentially the same. S, Mg, P and K in RBC of smokers were higher than in male non-smokers, and Ca was lower. S and Ca in serum of smokers were significantly lower than in male non-smokers. P in smokers was higher than in non-smokers. P in RBC may possibly activate the reflux of Mg into RBC and may suppress that of Ca. In smokers, the correlation coefficient (gamma) between Fe and Ca was r = -0.68 in RBC, and r = 0.76 in serum. Also gamma between P and Mg was r = 0.4 in RBC and r = -0.48 in serum. Thus Fe in RBC may suppress the reflux of Ca. Mg and Ca in serum of females were significantly higher than in male non-smokers. Ca in RBC of females was significantly higher than in male non-smokers, and P, K and Fe were significantly lower. The product of Ca and P in RBC of females was lower than in male non-smokers, while in serum it was higher. gamma between P and Ca, and between Fe and Ca in RBC of females were negative, while they were positive in male non-smokers. The correlations in female RBC had the same pattern as in male smokers.

A method for determining the distribution of fluoride, calcium and phosphorus in human dental plaque and the effect of a single in vivo fluoride rinse.

A new sampling method, capable of sampling plaque from its surface to its interior for quantitative studies, was modified to meet some of the requirements for the determination of the fluoride and mineral (Ca and P) profiles within dental plaque formed in vivo. Plaque samples were repeatedly collected from the same individual, using special devices, before a single fluoride rinse (900 parts/10(6) fluoride) and 10 min and 24 hr after rinse. The method allowed examination of fluoride, calcium and phosphorus distribution along the entire thickness of plaque. Fluoride content significantly increased throughout the sample 10 min after rinsing, indicating the fluoride had rapidly penetrated into the plaque. Although the elevated fluoride concentrations diminished almost to baseline with 24 hr, a high correlation was found between fluoride and minerals in each plaque fraction. It is concluded that this technique will be useful for evaluating the fluoride and mineral behaviour in the saliva/plaque and plaque/enamel interfaces, and the anti caries efficacy of fluoride applications.

Fluoride profiles in dental calculus from Japanese, Chinese and British residents.

Whether the fluoride concentrations and profiles differ in human dental calculus obtained from different countries was investigated. A total of 203 dental calculus deposits on 203 permanent teeth from residents (mean age, 52.1 years) of Nagoya (Japan), Shanghai (China), Leeds (Great Britain) and the Wuhan mountainous area (China, fluoridated area) were analysed. An abrasive microsampling procedure was used to examine fluoride distribution. There were five types of fluoride profiles in dental calculus in each area/country (designated types L, J, U, T, W). In supragingival calculus, type L (highest in the outermost layers) and type J (highest in the innermost layers) both had significantly higher values than type U (high in the surface and innermost layers) but were relatively identical. In subgingival calculus, type W (high in the outermost, mid and innermost layers) was characteristics. Calculus from the Wuhan mountainous area (fluoridated) had the highest fluoride concentration, followed by Leeds (non-fluoridated), and Nagoya and Shanghai (non-fluoridated) calculus had the lowest. Fluoride concentrations in supragingival calculus were higher in teeth extracted because of periodontal diseases than dental caries. It is concluded that fluoride concentrations and distribution in dental calculus differ from country to country, probably due to different fluoride environments.

Genetic variability of the common nod gene in soybean bradyrhizobia isolated in Thailand and Japan.

To determine the taxonomic relationship between Thai soybean bradyrhizobia and soybean bradyrhizobia from other regions, a total of 62 Bradyrhizobium strains were isolated in Thailand. The genetic diversity of the strains was examined with reference to 46 Japanese and 15 USDA strains. The degree of sequence divergence in and around common nod gene regions of the 123 strains was estimated by restriction fragment length polymorphism analysis using the Bradyrhizobium japonicum USDA 110 common nodDYABC gene probe. The phylogenetic grouping of the strains resulted in four major clusters. Cluster 1 comprised the Japanese and USDA strains, which originated in temperate regions, whereas clusters 3 and 4 comprised the tropical Thai strains. Cluster 1 strains comprised the DNA homology groups I and Ia, and hence, were classified as B. japonicum. Cluster 2 strains were in the DNA homology group II, and hence, were classified as Bradyrhizobium elkanii. Clusters 3 and 4 strains, however, did not correspond to any known DNA homology groups. These results indicate that Thai soybean bradyrhizobia are distantly related to B. japonicum and B. elkanii.

Follow-up study of thyroid function in polytransfused thalassemic patients.

The aim of our investigation was to evaluate thyroid function by a follow-up study in 45 polytransfused thalassemic patients, since endocrine abnormalities are frequent consequences of iron overload in thalassemia major. Significant changes of thyroid function have been revealed in the time elapsing the observation, despite unchanged haematological parameters; at the end of the present study five patients were affected by overt hypothyroidism and 15 patients by subclinical hypothyroidism. Ultrasound thyroid volume in 13 randomly selected patients was greatly reduced, while thyroid Magnetic Resonance Imaging (MRI) was not able to detect tissue alterations. Inversely, liver MRI was markedly reduced in 14 patients and negatively related to ferritine levels (P< 0.01). We conclude that polytransfused thalassemics are frequently affected by thyroid disfunction; haepatic haemosiderosis due to iron overload seems influence hormonal peripheral metabolism, although the patients display a moderate compliance with iron chelation therapy. Therefore, periodic thyroid investigation should be carried out in thalassemic subjects in order to detect patients who need hormone replacement therapy.

[Basic and clinical studies on biapenem (L-627) in obstetrics and gynecology].

UNLABELLED: We investigated biapenem (BIPM, L-627) a newly carbapenem antibiotic, for its antibacterial activity, tissue penetration, clinical efficacy and bacteriological effect in obstetric and gynecological infections, and obtained the following results. 1. Antibacterial activity: MICs of L-627 against 149 strains isolated from 80 patients in this clinical trial were examined and compared with those of imipenem (IPM) and ceftazidime (CAZ). The MIC50 and MIC90 of L-627 against the isolates were 0.2 and 12.5 micrograms/ml, respectively. Those of IPM were 0.2 and 6.25 micrograms/ml, respectively. The antibacterial activity of L-627 was quite similar to that of IPM, and was superior to that of CAZ. 2. Tissue and retroperitoneal fluid penetration: The peak levels in venous and uterine arterial sera were 24.0 and 26.2 micrograms/ml, respectively, after 300 mg drip infusion. The peak levels in the uterine or adnexal tissues were 2.39-9.60 micrograms/g, and 0.2 microgram/g of L-627 was detected at 275 minutes after administration. Peak levels in retroperitoneal fluid were 8.7 +/- 1.7 micrograms/ml at 1 hour after the completion of 30 minutes drip infusion (300 mg) and 7.9 +/- 0.2 micrograms/ml at 30 minutes after 300 mg 60 minutes drip infusion (300 mg). These levels expected the MICs against main pathogenic organisms. 3. CLINICAL RESULTS: L-627 was given to the following 144 patients (No. of analytical subjects) at a daily dose of 0.3-1.2 g for 2-13 days: intrauterine infections (54), adnexitis (36), parametritis (17), pelvic peritonitis (27), bartholins abscess (6) and other infections (4). The clinical efficacy was 93.1% (134/144) and the eradication rate against isolated organisms was 88.7% (110/124). Side effects were observed in 2 patients: eruption (1) and vomiting with numbness of the tongue (1). Abnormal change in laboratory test results included increase in eosinophils in 1, increase in GOT, GPT and gamma-GTP in 1 and increase in GPT and A1-P in 1, but all of these abnormalities were very mild and withdrawal of the drug was not required. Our results suggest that this drug is useful in the treatment of gynecological infections.

Effects of short-term high-dose testosterone propionate administration on medium molecular-weight proteins of human seminal plasma.

Effects of short-term high-dose testosterone propionate treatment on medium molecular-weight proteins (lactoferrin, albumin, prostatic acid phosphatase, prostate specific antigen) and on zinc and fructose levels were investigated in the seminal plasma of seven normal volunteers. A significant reduction in levels of prostatic-acid phosphatase, zinc and, to a lesser degree, prostate-specific antigen, lactoferrin and fructose was observed on the 14th day of androgen treatment, concomitantly with the maximal increase in free androgen-circulating levels. The data obtained suggest that testosterone administration may induce a reduction in the sex accessory-gland secretion. Indeed, this effect tends to disappear with withdrawal of hormone treatment. Therefore, the authors suggest a close follow-up of prostatic and vesicular function during the long-term high-dose testosterone intake, used frequently as anabolic treatment by athletes and body builders.

[Immunological and physicochemical properties of Cry j II, the second major allergen of Japanese cedar pollen (Cryptomeria japonica)].

Cry j II, the second major allergen of Japanese cedar (sugi, Cryptomeria japonica) pollen was examined for the allergenicity by intradermal test and RAST. Nineteen of the 25 allergic patients examined, showed positive reaction to the Cry j II. Contents of Cry j II in the extracts of the pollen collected in various regions from 1977 to 1991 showed yearly variation ranging from 2.9 to 14 mg/100 g pollen, whereas the amount of Cry j I in the extract was comparatively stable at about 35 mg/100 g pollen. Physicochemical treatments of Cry j I and Cry j II suggested that specific human IgE antibodies and some mAbs bind to conformational epitopes which are denatured and destroyed by certain treatments.

Epitopes on Cry j I and Cry j II for the human IgE antibodies cross-reactive between Cupressus sempervirens and Cryptomeria japonica pollen.

Forty sea from French patients allergic to Cupressus sempervirens pollen were tested for cross-reactivities against Cry j I, Cry j II (major allergens of Cryptomeria japonica pollen) and other pollen allergens from botanically related plants. Seventy-three per cent of the sera reacted with either Cry j I or Cry j II, or with both of them. These IgE cross-reactions were blocked effectively by mAb 046 (anti-Cry j I) or N26, T27 (anti-Cry j II), and weakly by mAbs 052, 027 and 026 (anti-Cry j I). Furthermore, the IgE antibodies in two sera, #40 and #11, bound to peptide fractions obtained from enzyme-digested Cry j I, and mAb 027 could also bind to the fractions. Analyses of the amino acid sequences of the peptides revealed that reactive peptides contained "NGNATPQLTKNAGVLTCSLSKR" sequence and the third residue N3 was glycosylated, however, when the N3 was not glycosylated, the IgE antibodies did not react, but mAb 027 could. The glycosylation of the N3 might be required for IgE-binding to the peptides. Sugar component on the N3 residue was found to be 0.4 mol galactose, 1.3 mol mannose, 0.8 mol fucose and 2.0 mol N-acetyl-glucosamine. Cross-reactivities against other pollen allergens from botanically related plants were found in most of the sera. However, many of these reactivities were detected by sandwich ELISA but not by an ELISA using allergen-coated plates, indicating that it is important to select an appropriate ELISA procedure in order to detect an allergen or an IgE antibody to an allergen.

Antigenic analyses of Sugi basic protein by monoclonal antibodies: I. Distribution and characterization of B-cell-tropic epitopes of Cry j I molecules.

Using 23 monoclonal antibodies raised against Sugi basic protein (SBP, major allergen of Japanese cedar pollen), composed of Cry j I and Cry j II, analyses of B-cell-tropic epitopes of Cry j I were performed. The following results were obtained. (1) As far as the mAbs were used, no major cross-reactive determinants were detected between Cry j I and Cry j II molecules. (2) 21 of the 23 mAbs were specific for Cry j I, and the anti-Cry j II mAbs were classified into four groups by their fine specificities, suggesting that Cry j I bears at least four antigenic determinant regions. (3) Cry j I molecules were found to take a monomeric form in solution and to display no repeating antigenic epitopes on their surfaces. (4) Some of the determinants seemed to be located in the interior of a Cry j I molecule, and when the Ag is coated on a plastic plate, the determinants become exposed on its surface. (5) Binding of human IgE antibodies to Cry j I and Cry j II was blocked by some of the obtained mAbs, suggesting that these epitopes recognized by the mAbs might have an important role in human allergic response against the cedar pollen.

Antigenic analyses of Sugi basic protein by monoclonal antibodies: II. Detection of immunoreactive fragments in enzyme-cleaved Cry j I.

The 4 anti-Cry j I mAbs showing an epitope specificity different from each other, 046, 029, 026 and 027, were selected to analyze the structure of the antigenic determinant for each mAb on a Cry j I molecule. Immunoreactive fragments in enzyme-cleaved Cry j I were detected by means of the adsorption on the mAb column and of the binding to the mAbs on Elisa. The mAb 026 was found to be reactive to the fragments containing a Cry j I N-terminal region obtained by V8 protease or pepsin digestion, but not to those by lysylendopeptidase digestion. The mAb 027 was found to be capable of binding to the fragments containing a linear structure of Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser-Leu-Ser-Lys, which were generated by V8 protease, lysylendopeptidase or pepsin digestion. Furthermore, the synthetic peptide Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser- Leu-Ser-Lys-Arg could bind to 027, but not to 026, and could inhibit the binding of 027 to Cry j I or to its immunoreactive fragments. No fragments capable of reacting to the mAbs 046 and 029 could be found in this study, suggesting that 046 and 029 recognize a conformationally constituted epitope of Cry j I molecule which is destroyed by enzymatic cleavage. The epitope recognized by the mAbs 027 or 026 was found to be located in conformationally hidden parts of the molecule which was exposed to react to the mAbs only after the physicochemical or enzymatic treatment.

Identification of the second major allergen of Japanese cedar pollen.

We isolated and characterized the second major allergen (Cry j II) from Japanese cedar pollen. We found that most patients with this pollinosis had IgE antibody to this protein in addition to IgE antibody to Cry j I; however, some sera reacted only with Cry j I or Cry j II. IgE-ELISA inhibition studies revealed that Cry j I and Cry j II had no cross-allergenicity. Cry j II did not react with anti-Cry j I monoclonal antibodies. In SDS-PAGE under a non-reducing condition, Cry j II showed a band at the 37 kDa position, compared with the 45-50 kDa bands of Cry j I. N-terminal amino acid sequence of Cry j II was completely different from that of Cry j I.

Participation of the lower brain stem in induction of preovulatory gonadotropin surges in female rats.

The role of the lower brain stem in controlling preovulatory gonadotropin surges was investigated in female rats under acute experimental conditions. Electrolytic lesions or diethyldithiocarbamate implantations in the ventrolateral part of the medulla oblongata (VLMO), which were carried out at 1100-1330 h on the day of proestrus, resulted in a blockade of the preovulatory surges of LH, FSH and PRL as well as subsequent ovulation. Such treatments in the dorsomedial part of the medulla oblongata did not affect gonadotropin surges or ovulation. By means of electrolytic lesions in the VLMO, norepinephrine concentrations were significantly reduced in the preoptic-anterior hypothalamic area at 1700-1800 h on proestrus, though they did not change in the mid-posterior hypothalamus. Electrochemical stimulations of the suprachiasmatic part of the preoptic area or norepinephrine injections into the third ventricle at 1400-1500 h on proestrus in animals with VLMO lesions succeeded in induce gonadotropin surges and ovulation. These results suggest that the lower brain stem is involved in the induction of preovulatory gonadotropin surges and that the process may be mediated by the ascending noradrenergic system which originates in the VLMO.