Therapeutic efficacy of Kangen-karyu against H2O2-induced premature senescence.

The anti-aging potential of Kangen-karyu extract was investigated using the mechanisms of the cellular aging model of stress-induced premature senescence (SIPS) in TIG-1 human fibroblasts. SIPS was induced by a sublethal dose of H2O2 and chronic oxidative stress with repeat treatment of low-dose H2O2. Reactive oxygen species generation, lipid peroxidation, and senescence-associated beta-galactosidase activity were elevated in TIG-1 cells under SIPS induced by H2O2. However, Kangen-karyu extract led to significant declines in these parameters, suggesting its role in ameliorating oxidative stress-related aging. It was also observed that SIPS due to H2O2 treatment led to the loss of cell viability, whereas Kangen-karyu extract improved cell viability by attenuating H2O2-induced oxidative damage. TIG-1 cells under the condition of SIPS caused by sublethal and chronic low doses of H2O2 showed nuclear factor-kappaB (NF-kappaB) translocation to the nucleus from the cytosol, while Kangen-karyu extract inhibited NF-kappaB nuclear translocation, implying that Kangen-karyu extract could exert an anti-aging effect through NF-kappaB modulation. In addition, treatment with Kangen-karyu extract under H2O2-induced chronic oxidative stress normalized the cell cycle by reducing the number of cells in the G0/G1 phase and elevating the proportion of those in the S phase, indicating the role of Kangen-karyu extract in cell cycle regulation. On the other hand, Kangen-karyu extract did not exert such an effect on cell cycle regulation under acute oxidative stress induced by sublethal H2O2. Furthermore, treatment with Kangen-karyu extract prolonged the lifespan of TIG-1 cells under SIPS. The present study suggests that Kangen-karyu might play a therapeutic role against the aging process caused by oxidative stress.

Estradiol-antagonistic activity of phenolic compounds from leguminous plants.

Natural flavonoids are currently receiving much attention because of their estrogenic and antiestrogenic properties. Six isoflavones (isoprunetin, isoprunetin 7-O-beta-D-glucopyranoside, isoprunetin 4',7-di-O-beta-D-glucopyranoside, genistein, genistein 7-O-beta-D-glucopyranoside, daidzein), four flavones (luteolin, luteolin 7-O-beta-D-glucopyranoside, luteolin 4'-O-beta-D-glucopyranoside, licoflavone C), isolated from Genista morisii and G. ephedroides (two Leguminosae plants of the Mediterranean area) together with two structurally related pterocarpans, bitucarpin A and erybraedyn C, isolated from Bituminaria bituminosa (Leguminosae), were tested for the antagonist activity by a yeast based estrogen receptor assay (Saccharomyces cerevisiae RMY326 ER-ERE). Most compounds inhibited the estradiol-induced transcriptional activity in a concentration dependent manner. In particular, for the flavone luteolin 77% inhibition of the induced beta-galactosidase activity was observed. Interestingly, licoflavone C exhibited a dose-dependent antagonistic activity at concentrations up to 10(-4) M, but stimulated beta-galactosidase expression at higher concentrations resulting in a U-shaped-like dose-response curve.

Cytotoxicity and gene induction by some essential oils in the yeast Saccharomyces cerevisiae.

In order to get an insight into the possible genotoxicity of essential oils (EOs) used in traditional pharmacological applications we tested five different oils extracted from the medicinal plants Origanum compactum, Coriandrum sativum, Artemisia herba alba, Cinnamomum camphora (Ravintsara aromatica) and Helichrysum italicum (Calendula officinalis) for genotoxic effects using the yeast Saccharomyces cerevisiae. Clear cytotoxic effects were observed in the diploid yeast strain D7, with the cells being more sensitive to EOs in exponential than in stationary growth phase. The cytotoxicity decreased in the following order: Origanum compactum>Coriandrum sativum>Artemisia herba alba>Cinnamomum camphora>Helichrysum italicum. In the same order, all EOs, except that derived from Helichrysum italicum, clearly induced cytoplasmic petite mutations indicating damage to mitochondrial DNA. However, no nuclear genetic events such as point mutations or mitotic intragenic or intergenic recombination were induced. The capacity of EOs to induce nuclear DNA damage-responsive genes was tested using suitable Lac-Z fusion strains for RNR3 and RAD51, which are genes involved in DNA metabolism and DNA repair, respectively. At equitoxic doses, all EOs demonstrated significant gene induction, approximately the same as that caused by hydrogen peroxide, but much lower than that caused by methyl methanesulfonate (MMS). EOs affect mitochondrial structure and function and can stimulate the transcriptional expression of DNA damage-responsive genes. The induction of mitochondrial damage by EOs appears to be closely linked to overall cellular cytotoxicity and appears to mask the occurrence of nuclear genetic events. EO-induced cytotoxicity involves oxidative stress, as is evident from the protection observed in the presence of ROS inhibitors such as glutathione, catalase or the iron-chelating agent deferoxamine.

Effects of 4 weeks of creatine supplementation in junior swimmers on freestyle sprint and swim bench performance.

To determine whether 4 weeks of oral creatine (Cr) supplementation could enhance single freestyle sprint and swim bench performance in experienced competitive junior swimmers, 10 young men and 10 young women (x age = 16.4 +/- 1.8 years) participated in a 27-day supplementation period and pre- and posttesting sessions. In session 1 (presupplementation testing), subjects swam one 50-m freestyle and then (after approximately 5 minutes of active recovery) one 100-m freestyle at maximum speed. Blood lactate was measured before and 1 minute after each swim trial. Forty-eight hours later, height, mass, and the sum of 6 skinfolds were recorded, and a Biokinetic Swim Bench total work output test (2 x 30-second trials, with a 10-minute passive recovery in between) was undertaken. After the pretests were completed, participants were divided into 2 groups (n = 10, Cr; and n = 10, placebo) by means of matched pairs on the basis of gender and 50-m swim times. A Cr loading phase of 20 g x d(-1) for 5 days was then instituted, followed by a maintenance phase of 5 g x d(-1) for 22 days. Postsupplementation testing replicated the presupplementation tests. Four weeks of Cr supplementation did not influence single sprint performance in the pool or body mass and composition. However, 30-second swim bench total work scores for trial 1 and trial 2 increased after Cr (p < 0.05) but not placebo ingestion. Postexercise blood lactate values were not different after supplementation for the 50- and 100-m sprint trials either within or between groups. It was concluded that 4 weeks of Cr supplementation did not significantly improve single sprint performance in competitive junior swimmers, but it did enhance swim bench test performance.

Employment of the human estrogen receptor beta ligand-binding domain and co-activator SRC1 nuclear receptor-binding domain for the construction of a yeast two-hybrid detection system for endocrine disrupters.

To screen a wide variety of chemicals for endocrine disrupters, and to develop an effective microbial degradation system for them, a good system is needed for the rapid and accurate evaluation of the endocrine-disrupting activities of suspected chemicals and their degradation products. We constructed two-hybrid systems that co-express the Gal4p DNA binding domain/ligand-binding domain of human estrogen receptor (hER) alpha or beta and the Gal4p transactivation domain/nuclear receptor-binding domain of co-activator SRC1, TIF2, or AIB1 in Saccharomyces cerevisiae with a chromosome-integrated lacZ reporter gene under the control of Gal4p-binding sites. We found that the combination of the hERbeta ligand-binding domain and SRC1 nuclear receptor-binding domain was most effective for the xenoestrogen-dependent induction of reporter activity. The extent of transcriptional activation by known xenoestrogens and phytoestrogens was found to correlate well with their estrogenic activities as measured by the previous system with rat ERalpha. This system detects estrogenic activity in some chemicals that have not been suspected of being positive. We also applied this assay system to test the microbial degradation products of gamma-hexachlorocyclohexane (gamma-HCH) by Sphingomonas paucimobilis. Among the gamma-HCH metabolites, 2,5-dichlorohydroquinone and chlorohydroquinone had estrogenic activities similar to the original chemical, while hydroquinone, a later stage metabolite, showed no activity, suggesting the necessity of evaluating intermediate metabolites in microbial degradation systems.

Human dermal fibroblasts escape from the long-term phenocopy of senescence induced by psoralen photoactivation.

We have previously shown that following psoralen photoactivation (PUVA treatment) human dermal fibroblasts undergo long-term growth arrest as well as morphological and functional changes reminiscent of cellular senescence [ 1 ]. In the absence of molecular data on what constitutes normal senescence, it has been difficult to decide whether these PUVA-induced changes reflect cellular senescence or rather a mimic thereof. We herein report that PUVA-induced growth arrest, the senescent phenotype with long-term induction of senescence-associated beta-galactosidase, as well as increased expression of matrix metalloprotease-1 are fully reversible at days 100 to 130 post PUVA treatment in four independently tested fibroblast strains. The late returning growth capacity in PUVA-treated fibroblasts is not due to immortalization, as shown by continued lack of telomerase activity, accelerated telomere shortening, and a decrease in overall growth rates in fibroblasts in their regrowing phase post PUVA treatment. Lack of anchorage-independent growth additionally suggests that the cells are also not tumorigenically transformed. Collectively, our data suggest that PUVA-induced changes do not fully reflect replicative senescence but rather represent a long-term transient phenocopy of senescence. The model reported here is particularly suited to elucidating mechanisms underlying long-term transient growth arrest, the related functional changes, and the release of cells thereof.

Photodynamic therapy-mediated oxidative stress as a molecular switch for the temporal expression of genes ligated to the human heat shock promoter.

Oxidative stress associated with photodynamic therapy (PDT) is a transcriptional inducer of genes encoding stress proteins, including those belonging to the heat shock protein (hsp) family. The efficiency of PDT to function as a molecular switch by initiating expression of heterologous genes ligated to the human hsp promoter was examined in the present study. Selective and temporal reporter gene expression was documented after PDT in mouse radiation-induced fibrosarcoma cells stably transfected with recombinant vectors containing an hsp promoter ligated to either the lac-z or CAT reporter genes and in transfected radiation-induced fibrosarcoma tumors grown in C3H mice. Hyperthermia treatments were included as a positive control for all experiments. Expression vectors containing either human p53 or tumor necrosis factor (TNF)-alpha cDNA under the control of an hsp promoter were also constructed and evaluated. A p53 null and TNF-alpha-resistant human ovarian carcinoma (SKOV-3) cell line was stably transfected with either the p53 or TNF-alpha constructs. Inducible expression and function of p53 as well as inducible expression, secretion, and biological activity of TNF-alpha were documented after PDT or hyperthermia in transfected SKOV cells. These results demonstrate that PDT-mediated oxidative stress can function as a molecular switch for the selective and temporal expression of heterologous genes in tumor cells containing expression vectors under the control of an hsp promoter.

Transcriptional regulation of the pas gene of enterohemorrhagic Escherichia coli.

The Pas protein plays a key role in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC), being required for the secretion of the Esp proteins. Here, the transcriptional regulation of the pas gene was analyzed through the construction of a pas::lacZ translational fusion. When bacteria were grown in Luria Bertani medium or tissue culture medium supplemented with HEPES, a bimodal activation curve was observed. The early phase of induction was not significantly modified by the incubation temperature (either 25 or 37 degrees C), whereas the second phase, which overlaps with the late exponential growth phase, was enhanced at 37 degrees C. The early phase was also stimulated by growth on tissue culture medium and by the addition of Ca(2+), Mn(2+)or Mg(2+) to the M9-glucose minimal medium. Primer extension analysis showed the presence of two major starts of transcription, which were located 58 and 60 bp upstream of the ATG-start codon of the Pas protein, respectively. Although these sites are very close to each other, the transcripts produced during the early induction phase mainly start on the -60 position, whereas the -58 start was activated during the second induction phase.

Anti-diarrhoeal and gastro-intestinal potentials of the aqueous extract of Phyllanthus amarus (Euphorbiaceae).

The anti-diarrhoeal and gastro-intestinal protective potentials of aqueous extract of leaves of Phyllanthus amarus were investigated in mice. Graded doses of the aqueous extract (100-800 mg/kg) administered orally produced a dose-related inhibition of gut meal travel distance in normal mice. The highest intestinal transit inhibition of 31.65% was obtained with 400 mg/kg. In castor oil induced diarrhoea in mice, P. amarus extract (400 mg/kg) delayed the onset of diarrhoea, reduced frequency of defecation and reduced gut meal travel distance significantly resulting in intestinal transit inhibition of 79.94% compared to 86.92% produced by morphine (100 mg/kg). In addition, the activities of some intestinal mucosa enzymes (maltase, sucrase, lactase and alkaline phosphatase) in mice pretreated with extract before castor oil were not as severely depressed as those in the control (castor oil treated mice). Phytochemical screening revealed the presence of many secondary metabolites. The results are discussed with a view to establishing the basis of the use of this plant in traditional medicine for treatment of diarrhoea and other gastrointestinal disorders.

Effects of two highly monounsaturated oils on lipid composition and enzyme activities in rat jejunum.

The effects of two monounsaturated fatty acid (MUFA) oils, olive oil (OO) and high-oleic sunflower oil (HOSO), with high content in oleic acid but differing in their non-fatty acid fraction, on brush-border membrane (BBM) lipid composition and fluidity and on mucosal enzyme activities of rat jejunum were studied. Animals were given semipurified diet with linoleic acid to prevent essential fatty acid deficiency (control group) or semipurified diet containing 10% of either OO or HOSO for 12 weeks. There was a significant decrease in the content of jejunal BBM phospholipids together with an increase in the level of free cholesterol in both oil-fed rats, when compared to control group. Although the increase in the BBM free cholesterol level was not statistically significant in HOSO-fed rats, a significant decrease in the phospholipid/free cholesterol ratio was found in both OO and HOSO-fed animals compared to control group. Rat jejunal BBM had a high level of free fatty acids which was increased in BBM isolated from OO and HOSO-fed animals. There was no statistical significant difference in the phospholipid distribution between the control and the OO group. However, HOSO-fed animals showed the lowest level of phosphatidylethanolamine together with the highest phosphatidylcholine content and the phosphatidylcholine/sphingomyelin ratio. The fatty acid pattern of jejunal BBM lipids was modified according to the major fatty acids in the oils. There was a decrease in both stearic acid (18:0) and linoleic acid (18:2 n-6), together with an increase in oleic acid (18:1 n-9) in jenunal BBM isolated from both oil experimental groups. All these results were accompanied by a significant increase in the BBM fluidity (as assessed by steady-state fluorescence polarization of diphenylhexatriene) isolated from oil-fed rat, when compared to control group. OO and HOSO-fed animals had the lowest activities of sucrase and maltase, while alkaline phosphatase activity only was decreased in HOSO-fed animals. The specific activity of maltase was not modified in any experimental rats. In summary, both MUFA oils induced similar effects on jejunal BBM lipid composition, fluidity, sucrase, maltase and lactase activities. Furthermore, HOSO intake resulted in a lowest alkaline phosphatase activity which was accompanied by changes in individual phospholipid composition. All these results suggest that effects of MUFA oils on jejunal BBM lipid composition and hydrolase activities are most likely due to the presence of high content of oleic acid rather than other components contained in the non-fatty acid of olive oil.

Effect of colostrinin, an immunomodulatory proline-rich polypeptide from ovine colostrum, on sialidase and beta-galactosidase activities in murine thymocytes.

Colostrinin: a proline-rich polypeptide (PRP) from ovine colostrum and its nonapeptide active fragment (NP) induce maturation and differentiation of murine thymocytes, formation of helper cells from PNAhigh thymocytes and cytotoxic T cells from PNAlow thymocytes. These processes are accompanied by changes in expression of receptors for peanut agglutinin (PNA), PNAhigh thymocytes were transformed into PNAlow cells, and vice versa. It was shown, in various laboratories, that sialyltransferases are involved in the transformation of PNAhigh thymocytes into PNAlow cells. To find out whether the expression of receptors for PNA on murine thymocytes might also be influenced by other enzymes, we decided to study the effect of PRP and NP on sialidase and beta-galactosidase activities in these cells. The results obtained showed that the most of sialidase activity of murine thymocytes is present in the plasma membrane compartments. Both thymocyte subpopulations PNAhigh and PNAlow, showed similar sialidase activity, which was not affected either by PRP or NP. In contrast to sialidases, most of beta-galactosidase activity was present in the cytosol. PNAhigh, thymocytes showed a higher beta-galactosidase activity than PNAlow cells. Incubation of immature, PNAhigh, thymocytes with PRP or NP enhanced the beta-galactosidase activity in these cells. The presented results suggest that sialidases seem not to be involved in modulation of surface sialic acid content during murine thymocyte maturation. On the other hand, stimulation of activity of beta-galactosidase in PNAhigh, immature thymocytes by PRP and NP suggests that beta-galactosidase in murine thymocytes might be involved in transformation of PNAhigh into PNAlow cells.

Nucleotide supplements alter proliferation and differentiation of cultured human (Caco-2) and rat (IEC-6) intestinal epithelial cells.

The effect of exogenous nucleotides on the proliferation and differentiation of enterocytes was comparatively studied using a human colon tumor cell line (Caco-2) and a normal rat small intestinal crypt cell line (IEC-6). Caco-2 cells exhibited more active endogenous nucleic acid metabolism than did IEC-6 cells, as evidenced by greater cellular pools of nucleotides and their metabolites. To determine the supplemental nutritional effect of nucleotides, a mixture containing equal amounts (10 mg/L) of AMP, CMP, IMP, GMP and UMP was added to the culture medium. The results showed that a nucleotide supplement under normal culture conditions did not affect proliferation and differentiation of Caco-2 cells. In contrast, nucleotide supplements under normal culture conditions promoted proliferation of IEC-6 cells. The addition of nucleotides to the culture medium also enhanced differentiation of IEC-6 cells when grown on an extracellular matrix (Matrigel). Furthermore, when glutamine levels were less than optimal (nutritional stress conditions), nucleotide supplements enhanced growth and maturation of both cell lines. We conclude that the de novo biosynthesis of nucleotides is sufficient to support proliferation of tumor Caco-2 cells but not of the normal crypt IEC-6 cells. Thus, nucleotide supplements may enhance normal enterocyte growth and maturation as well as spare the need for exogenous glutamine in cell maintenance and development.