Critical determinants of estrogen receptor -mediated agonist activity

Exogenous ligands that bind to the estrogen receptor (ER) exhibit unique pharmacologies distinct from that observed with the endogenous hormone, 17β-estradiol (ED. Differential activity among ER ligands has been observed at the level of receptor binding, promoter interaction and transcriptional activation. Furthermore, xenoestrogens can display tissue-specific agonist activity on the cellular level, functioning as an agonist in one tissue and as an antagonist in another. That the same ligand, functioning through the same receptor, can produce differing agonist responses on the cellular level indicates that there are tissue-specific determinants of agonist activity. In these studies critical molecular determinants of agonist activity were characterized for several cell types. In the normal and neoplastic myometrium a proliferative response was dependent upon activation of AF2 of the ER, functioning as a determinant of agonism in this cell type. Progesterone receptor (PR) ligands transdominantly suppressed ER-mediated transcription and proliferation in uterine leiomyoma cells, indicating that ER/PR cross-talk can modulate agonist activity in a myometrial cell background. In the breast, the agonist response to ER ligands was investigated by employing a functional genomics approach to generate gene expression profiles. Treatment of breast cancer cells with the selective estrogen receptor modulator tamoxifen largely recapitulated the expression profile induced by treatment with the agonist E2, despite the well-characterized antiproliferative effects produced by tamoxifen in this cell type. While the expression of many genes involved in regulating cell cycle progression, including fos, myc, cdc25a, stk15 and cyclin A, were induced by both E2 and tamoxifen in breast cells, treatment with the agonist E2 specifically induced the expression of cyclin D1, fra-1 , and uracil DNA glycosylase. These results suggest that the inability of tamoxifen to transactivate expression of only a few key genes, functioning as cellular gatekeepers, prevent tamoxifen-treated breast cells from entering the cell cycle. Thus, the expression of these agonist-specific marker genes is a potential determinant of agonist activity at the cellular level in the breast. Collectively, studies in the breast and uterine myometrium have identified several mechanisms whereby ER ligands modulate ER-mediated signaling and provide insights into the biology of tissue-specific agonist activity in hormone-responsive tissues. ^

Regulation of Bcl-2 expression by the product of the prostate apoptosis response gene -4, Par-4

Elevated expression levels of the bcl-2 proto-oncogene have been correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t (14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer, there being no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2 expressing, androgen-independent prostate cancer cell lines, by all trans-retinoic acid treatment but this did not occur in the androgen-dependent cell lines expressing low levels of Bcl-2. Changes in localization of Par-4, and an induction in the expression of WT1 protein accompanied the decrease in the Bcl-2 protein and transcript following all trans-retinoic acid treatment, in the androgen-independent prostate cancer cell line. In stable clones expressing ectopic Par-4 we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1 binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1 binding site on the bcl-2 promoter. ^

Impact of BCR -ABL on DNA repair

The BCR-ABL fusion gene is the molecular hallmark of Philadelphia-positive leukemias. Normal Bcr is a multifunctional protein, originally localized to the cytoplasm. It has serine kinase activity and has been implicated in cellular signal transduction. Recently, it has been reported that Bcr can interact with xeroderma pigmentosum group B (XPB/ERCC3)—a nuclear protein active in UV-induced DNA repair. Two major Bcr proteins (p160 Bcr and p130Bcr) have been characterized, and our preliminary results using metabolic labeling and immunoblotting demonstrated that, while both the p160 and p130 forms of Bcr localized to the cytoplasm, the p130 form (and to a lesser extent p160) could also be found in the nucleus. Furthermore, electron microscopy confirmed the presence of Bcr in the nucleus and demonstrated that this protein associates with metaphase chromatin as well as condensed interphase heterochromatin. Since serine kinases that associate with condensed DNA are often cell cycle regulatory, these observations suggested a novel role for nuclear Bcr in cell cycle regulation and/or DNA repair. However, cell cycle synchronization analysis did not demonstrate changes in levels of Bcr throughout the cell cycle. Therefore we hypothesized that BCR serves as a DNA repair gene, and its function is altered by formation of BCR-ABL. This hypothesis was investigated using cell lines stably transfected with the BCR-ABL gene, and their parental counterparts (MBA-1 vs. M07E and Bcr-AblT1 vs. 4A2+pZAP), and several DNA repair assays: the Comet assay, a radioinimunoassay for UV-induced cyclobutane pyrimidine dimers (CPDs), and clonogenic assays. Comet assays demonstrated that, after exposure to either ultraviolet (UV)-C (0.5 to 10.0 joules m −2) or to gamma radiation (200–1000 rads) there was greater efficiency of DNA repair in the BCR-ABL-transfected cells compared to their parental controls. Furthermore, after UVC-irradiation, there was less production of CPDs, and a more rapid disappearance of these adducts in BCR-ABL-bearing cells. UV survival, as reflected by clonogenic assays, was also greater in the BCR-ABL-transfected cells. Taken together, these results indicate that, in our systems, BCR-ABL confers resistance to UVC-induced damage in cells, and increases DNA repair efficiency in response to both UVC- and gamma-irradiation. ^

Structural and functional analysis of p84N5

Normal development and tissue homeostasis requires the carefully orchestrated balance between cell proliferation and cell death. Cell cycle checkpoints control the extent of cell proliferation. Cell death is coordinated through the activation of a cell suicide pathway that results in the morphologically recognizable form of death, apoptosis. Tumorigenesis requires that the balance between these two pathways be disrupted. The tumor suppressor protein Rb has not only been shown to be involved in the enforcement of cell cycle checkpoints, but has also been implicated in playing a role in the regulation of apoptosis. The manner in which Rb enforces cell cycle checkpoints has been well studied; however, its involvement in the regulation of apoptosis is still very unclear. p84N5 is a novel nuclear death domain containing protein that has been shown to interact with the N-terminus of Rb. The fact that it contains a death domain and the fact that it is nuclear localized possibly provides the first known mechanism for apoptotic signaling from the nucleus. The following study tested the hypothesis that the novel exclusively nuclear death domain containing protein p84N5 is an important mediator of programmed cell death and that its apoptotic function is reliant upon its nuclear localization and is regulated by unique functional domains within the p84N5 protein. We identified the p84N5 nuclear localization signal (NLS), eliminated it, and tested the functional significance of nuclear localization by using wild type and mutant sequences fused to EGFP-C1 (Clontech) to create wild type GFPN5 and subsequent mutants. The results of these assays demonstrated exclusive nuclear localization of GFPN5 is required for normal p84N5 induced apoptosis. We further conducted large-scale mutagenesis of the GFPN5 construct to identify a minimal region within p84N5 capable of interacting with Rb. We were able to identify a minimal sequence containing p84N5 amino acids 318 to 464 that was capable of interacting with Rb in co-immunoprecipitation assays. We continued by conducting a structural and functional analysis to identify the region or regions within p84N5 responsible for inducing apoptosis. Point mutations and small-scale deletions within the death domain of p84N5 lessened the effect but did not eliminate p84N5-induced cytotoxicity. Further analysis revealed that the minimal sequence of 318 to 464 of p84N5 was capable of inducing apoptosis to a similar degree as wild-type GFPN5 protein. Since amino acids 318 to 464 of p84N5 are capable of inducing apoptosis and interacting with Rb, we propose possible mechanisms whereby p84N5 may function in a Rb regulated manner. These results demonstrate that p84N5 induced apoptosis is reliant upon its nuclear localization and is regulated by unique functional domains within the p84N5 protein. ^

The role of platelet -derived growth factor (PDGF) signaling in gliomagenesis

Gliomas are primary central nervous system (CNS) neoplasms that are believed to arise from astrocytes, oligodendrocytes or their precursors. Gliomas can be classified into two major histopathological groups: oligodendroglial and astroglial tumors. The most malignant of the astroglial tumors is glioblastoma multiforme (GBM). A great deal of genetic and epigenetic alterations have been implicated in gliomagenesis. In particular, PDGF signaling is frequently over-activated in a large number of human gliomas. In order to gain insights into the biology of gliomas, we manage to model human gliomas in mice using a somatic gene transfer approach—RCAS/TVA system. In our previous study, combined activation of AKT and RAS pathways gave rise to glioblastomas from CNS progenitors. In the present study, we demonstrate that in vivo autocrine PDGF stimulation induces oligodendrogliomas and mixed oligoastrocytomas from CNS progenitors and differentiated astrocytes respectively. In culture autocrine PDGF stimulation dedifferentiates astrocytes into progenitor-like cells and blockade of PDGF signaling reverses these phenotypic changes. Experimental disruption of cell cycle arrest pathway, such as Ink4a-Arf loss, is not required for the initiation of PDGF-induced gliomagenesis; instead, this mutation contributes to the tumor progression by enhancing tumor malignancy and shortening tumor latency. P53 deficiency does not promote the PDGF-induced gliomagenesis. In addition, 1p and 19q, often deleted in human oligodendrogliomas, remain intact in these PDGF-induced gliomas. Therefore, our studies suggest that autocrine PDGF stimulation alone may be sufficient to induce gliomagenesis. In contrast to transient stimulation in vitro, constitutive PDGF stimulation activates neither AKT nor RAS/MAPK pathways during gliomagenesis. This results in the formation of oligodendrogliomas, instead of glioblastomas. Sustained activation of the AKT pathway converts PDGF-induced oligodendrogliomas into astrocytomas. Our studies suggest that constitutive PDGF stimulation is not equivalent to transient PDGF stimulation, and that a transition between oligodendroglial and astroglial tumors in humans may be possible, depending on additional alterations. In summary, PDGF signaling plays a pivotal role in gliomagenesis in the mouse, and its hyperactivity is capable of contributing to both oligodendroglial and astroglial tumorigenesis. ^

A role for p53 in sensing DNA damage and triggering apoptotic responses to anticancer agents

The p53 tumor suppressor protein plays a major role in cellular responses to anticancer agents that target DNA. DNA damage triggers the accumulation of p53, resulting in the transactivation of genes, which induce cell cycle arrest to allow for repair of the damaged DNA, or signal apoptosis. The exact role that p53 plays in sensing DNA damage and the functional consequences remain to be investigated. The main goal of this project was to determine if p53 is directly involved in sensing DNA damage induced by anticancer agents and in mediating down-stream cellular responses. This was tested in two experimental models of DNA damage: (1) DNA strand termination caused by anticancer nucleoside analogs and (2) oxidative DNA damage induced by reactive oxygen species (ROS). Mobility shift assays demonstrated that p53 and DNA-PK/Ku form a complex that binds DNA containing the anticancer nucleoside analog gemcitabine monophosphate in vitro. Binding of the p53-DNA-PK/Ku complex to the analog-containing DNA inhibited DNA strand elongation. Furthermore, treatment of cells with gemcitabine resulted in the induction of apoptosis, which was associated with the accumulation of p53 protein, its phosphorylation, and nuclear localization, suggesting the activation of p53 to trigger apoptosis following gemcitabine induced DNA strand termination. The role of p53 as a DNA damage sensor was further demonstrated in response to oxidative DNA damage. Protein pull-down assays demonstrated that p53 complexes with OGG1 and APE, and binds DNA containing the oxidized DNA base 8-oxoG. Importantly, p53 enhances the activities of APE and OGG1 in excising the 8-oxoG residue as shown by functional assays in vitro. This correlated with the more rapid removal of 8-oxoG from DNA in intact cells with wild-type p53 exposed to exogenous ROS stress. Interestingly, persistent exposure to ROS resulted in the accelerated onset of apoptosis in cells with wild-type p53 when compared to isogenic cells lacking p53. Apoptosis in p53+/+ cells was associated with accumulation and phosphorylation of p53 and its nuclear localization. Taken together, these results indicate that p53 plays a key role in sensing DNA damage induced by anticancer nucleoside analogs and ROS, and in triggering down-stream apoptotic responses. This study provides new mechanistic insights into the functions of p53 in cellular responses to anticancer agents. ^

Dysregulated CD40-NF-kappaB signaling in the pathophysiology of aggressive non -Hodgkin's lymphoma B cells

Non-Hodgkin's Lymphomas (NHL) are a group (>30) of important human lymphoid cancers that unlike other tumors today, are showing a marked increase in incidence. The lack of insight to the pathogenesis of B-cell NHL poses a significant problem in the early detection and effective treatment of these malignancies. This study shows that large B-cell lymphoma (LBCL) cells, the most common type of B-cell NHL (account for more than 30% of cases), have developed a novel mechanism for autonomous neoplastic B cell growth. We have identified that the key transcription factor NF-κB, is constitutively activated in LBCL cell lines and primary biopsy-derived LBCL cells, suggesting that they are autonomously activated, and do not require accessory T-cell signaling for cell growth and survival. Further studies have indicated that LBCL cells ectopically express an important T-cell associated co-mitogenic factor, CD154 (CD40 ligand), that is able to internally activate the CD401NF-κB pathway, through constitutive binding to its cognate receptor, CD40, on the lymphoma cell surface. CD40 activation triggers the formation of a “Signalosome” comprising virtually the entire canonical CD40/NF-κB signaling pathway that is anchored by CD40 in plasma membrane lipid rafts. The CD40 Signalosome is vulnerable to interdiction by antibody against CD40 that disrupts the Signalosome and induces cell death in the malignant cells. In addition to constitutive NF-κB activation, we have found that the nuclear factor of activated T cells (NFAT) transcription factor is also constitutively activated in LBCL cells. We have demonstrated that the constitutively active NFATc1 and c-rel members of the NFAT and NF-κB families of transcription factors, respectively, interact with each other, bind to the CD154 promoter, and synergistically activate CD154 gene transcription. Down-regulation of NFATc1 and c-rel with small interfering RNA inhibits CD154 gene transcription and lymphoma cell growth. Our findings suggest that continuous CD40 activation not only provides dysregulated proliferative stimuli for lymphoma cell growth and extended tumor cell survival, but also allows continuous regeneration of the CD40 ligand in the lymphoma cell and thereby recharges the system through a positive feedback mechanism. Targeting the CD40/NF-κB signaling pathway could provide potential therapeutic modalities for LBCL cells in the future. ^

Upregulation of VEGF through p70S6K in ErbB2 -mediated angiogenesis and inhibition by combined herceptin plus taxol therapy

ErbB2 overexpression in breast tumors increases metastasis, angiogenesis, and reduces survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, a spontaneous metastasis assay was performed using human breast cancer cells transfected with constitutively active ErbB2 kinase (V659E), an ErbB2 kinase-dead mutant (K753M), or vector control. Mice injected with V659E had increased metastasis and tumor microvessel density; and the increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro, which resulted from increased VEGF by increased protein synthesis. This appeared to be mediated through a PI3K, Akt, mTOR, p70S6K-signaling pathway. Furthermore, V659E xenografts had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with control. To validate the clinical relevance of these findings, human breast tumor samples were examined. Tumors overexpressing ErbB2 correlated with p70S6K phosphorylation and VEGF expression, which significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Together, ErbB2 increases VEGF expression by activating the p70S6K signaling pathway, which may serve as targets for antiangiogenic and antimetastatic therapies. ^ Herceptin is an anti-ErbB2 antibody that demonstrated anti-tumor function, especially in combination with other chemotherapies such as Taxol, in patients with ErbB2-overexpressing tumors. Since the repeated administration of low-dose chemotherapy endorsed an antiangiogenic effect in vitro, and Herceptin was shown to inhibit angiogenesis in tumor xenografts, I investigated whether combined Taxol plus Herceptin treatment inhibits ErbB2-mediated angiogenic responses more effectively. Mice with ErbB2-overexpressing xenografts were treated with control, Herceptin, Taxol, or combination Herceptin plus Taxol. Mice treated with the combination exhibited reduced tumor volumes, tumor microvessel densities, and lung metastasis; and ErbB2-overexpressing cells treated with the combination secreted less VEGF, and stimulated less endothelial cell migration. Furthermore, Akt phosphorylation contributed to VEGF upregulation and was most effectively reduced by combination treatment. ^ In summary, ErbB2 activates signaling to Akt and p70S6K leading to increased VEGF and angiogenesis. Combination Herceptin plus Taxol treatment most effectively inhibited ErbB2-mediated angiogenesis, resulting in pronounced tumoricidal effects, and may be mediated through reduction of phosphorylated Akt, a positive regulator in the p70S6K pathway. ^

Breast cancer -specific activation of topoisomerase IIalpha and its application in the tumor -targeting gene therapy

To ensure the success of systemic gene therapy, it is critical to enhance the tumor specificity and activity of the promoter. In the current study, we identified the breast cancer-specific activity of the topoisomerase IIα promoter. We further showed that cdk2 and cyclin A activate topoisomerase IIα promoter in a breast cancer-specific manner. An element containing an inverted CCAAT box (ICB) was shown to respond this signaling. When the ICB-harboring topoisomerase IIα minimal promoter was linked with an enhancer sequence from the cytomegalovirus immediate early gene promoter (CMV promoter), this composite promoter, CT90, exhibited activity comparable to or higher than the CMV promoter in breast cancer cells in vitro and in vivo, yet expresses much lower activity in normal cell lines and normal organs than the CMV promoter. A CT90-driven construct expressing BikDD, a potent pro-apoptotic gene, was shown to selectively kill breast cancer cells in vitro and to suppress mammary tumor development in an animal model of intravenously administrated, liposome-delivered gene therapy. Expression of BikDD was readily detectable in the tumors but not in the normal organs of CT90-BikDD-treated animals. Finally, we demonstrated that CT90-BikDD treatment potentially enhanced the sensitivity of breast cancer cells to chemotherapeutic agents, especially doxorubicin and taxol. The results indicate that liposomal CT90-BikDD is a novel and effective systemic breast cancer-targeting gene therapy, and its combination with chemotherapy may further improve the current adjuvant therapy for breast cancer. ^

Molecular mechanism of the relaxin signaling pathways: Activation of cAMP, PI3K, and PKCzeta

Relaxin is a polypeptide hormone that has diverse effects on reproductive and non-reproductive tissues. Relaxin activates the G-protein coupled receptors, LGR7 and LRG8. Early studies described increased cAMP and protein kinase A activity upon relaxin treatment, but cAMP accumulation alone could not account for all of the relaxin-mediated effects. We utilized the human monocyte cell line THP-1 to study the mechanism of relaxin-stimulated CAMP production. ^ Relaxin treatment in THP-1 cells produces a biphasic time course in cAMP accumulation, where the first peak appears as early as 1–2 minutes with a second peak at 10–20 minutes. Selective inhibitors for phosphoinositide 3-kinase (P13K), such as wortmannin and LY294002, show a dose-dependent inhibition of relaxin-stimulated cAMP accumulation, specific for the second peak of the relaxin time course. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 is mediated by the activity of phosphodiesterases. Furthermore, LY294002 blocks upregulation of vascular endothelial growth factor transcript levels by relaxin. ^ To further delineate relaxin signaling pathways, we searched for downstream targets of PI3K that could activate adenylyl cyclase (AC). Protein kinase C ζ (PKCζ) was a prime candidate because it activates types II and V AC. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin-induced cAMP production to the same degree as LY294002 (∼40%). Relaxin stimulates PKCζ translocation to the plasma membrane in THP-1, MCF-7, PHM1-31, and MMC cells, as shown by immunocytochemistry. PKCζ translocation is P13K-dependent and independent of cAMP production. Antisense PKCζ oligodeoxynucleotides (PKCζ-ODNs) deplete both PKCζ transcript and protein levels in THP-1 cells. PKCζ-ODNs abolish relaxin-mediated PKCζ translocation and inhibit relaxin stimulation of cAMP by 40%, as compared to mock and random ODN controls. Treatment with LY294002 in the presence of PKCζ-ODNs results in little further inhibition. Taken together, we present a novel role for PI3K and PKCζ in relaxin stimulation of cAMP and provide the first example of the PKCζ regulation of AC in an endogenous system. Furthermore, we have identified higher order complexes of AC isoforms and PKA anchoring proteins in attempts to explain the differential coupling of relaxin to cAMP and PI3K-signaling pathways in various cell types. ^

The biology of the cell surface,

Bibliography: p. 370-380.

Role of proline residues in the expression and function of the human noradrenaline transporter

The aim was to investigate the roles of proline residues in extracellular loop 2 (P172, P183, P188 and P209) and transmembrane domains 2, 5, 11 and 12 (P108, P270, P526, P551, P552 and P570) in determining noradrenaline transporter (NET) expression and function. Mutants of human NET with these residues mutated to alanine were pharmacologically characterized. Mutation of P108, P270 and P526 disrupted cell surface expression, from [H-3]nisoxetine binding and confocal microscopy data. Mutations of P526, P551 and P570 reduced transporter turnover (V-max of [H-3]noradrenaline uptake/B-max of [H-3]nisoxetine binding) by 1.5-1.7-fold compared with wild-type NET, so these residues might be involved in conformational changes associated with substrate translocation. Conversely, mutations of P172, P183, P188 and P209 increased V-max/B-max by 2-3-fold compared with wild-type, indicating that the presence of these proline residues limits turnover of the NET. The mutations had few effects on apparent affinities of substrates or affinities of inhibitors, except decreases in inhibitor affinities after mutations of the P270 and P570 residues, and increases after mutation of the P526 residue. Hence, proline residues in extracellular loop 2 and in transmembrane domains have a range of roles in determining expression and function of the NET.

Secretion of tumor necrosis factor by human leukocytes stimulated with shark cartilage

To assess the role of shark cartilage as an immune modulator, acid, salt-soluble, and phosphate-buffered saline extracts were prepared from three different commercial sources (SL, TL, FDC) of cartilage and used to stimulate human leukocytes in vitro. Duplicate leukocyte cultures were set up, each containing 50 $\mu$l of endotoxin-free extract, 200 $\mu$l of cell suspension (2.4-2.5 $\times$ 10$\sp5$ cells) and 100 $\mu$l of medium and incubated at 37$\sp\circ$C. Cultures stimulated with LPS (5 $\mu$g/ml) or medium served as the positive and negative controls, respectively. Culture supernatants were assayed for TNF$\alpha$ by ELISA. Cartilage extracts stimulated cells to release significant levels of TNF$\alpha$ (p $<$.005); the highest response was obtained with the acid extract of SL cartilage. In comparison, response to corresponding extracts of bovine cartilage was lower (p $<$.05). The stimulatory activity was reduced (85%) following proteolytic digestion, and lost when extract was heated (60$\sp\circ$C, 20 min) or treated with urea (6M), suggesting that the active component(s) is a protein. ^

Coronary heart disease risk factors among tri-ethnic students at Florida International University

The present study identified and compared Coronary Heart Disease (CHD) risk factors quantified as “CHD risk point standards” (CHDRPS) among tri-ethnic (White non-Hispanic [WNH], Hispanic [H], and Black non-Hispanic [BNH]) college students. All 300 tri-ethnic subjects completed the Cardiovascular Risk Assessment Instruments and had blood pressure readings recorded on three occasions. The Bioelectrical Impedance Analysis (BIA) was used to measure body composition. Students' knowledge of CHD risk factors was also measured. In addition, a 15 ml fasting blood sample was collected from 180 subjects and blood lipids and Homocysteine (tHcy) levels were measured. Data were analyzed by gender and ethnicity using one-way Analysis of Variance (ANOVA) with Bonferroni's pairwise mean comparison procedure, Pearson correlation, and Chi-square test with follow-up Bonferroni's Chi-square tests. ^ The mean score of CHDRPS for all subjects was 19.15 ± 6.79. Assigned to the CHD risk category, college students were below-average risk of developing CHD. Males scored significantly (p < 0.013) higher for CHD risk than females, and BNHs scored significantly (p < 0.033) higher than WNHs. High consumption of dietary fat saturated fat and cholesterol resulted in a high CHDRPS among H males and females and WNH females. High alcohol consumption resulted in a high CHDRPS among all subjects. Mean tHcy ± SD of all subjects was 6.33 ± 3. 15 μmol/L. Males had significantly (p < 0.001) higher tHcy than females. Black non-Hispanic females and H females had significantly (p < 0.003) lower tHcy than WNH females. Positive associations were found between tHcy levels and CHDRPS among females (p < 0.001), Hs (p < 0.001), H males (p < 0.049), H females (p < 0.009), and BNH females (p < 0.005). Significant positive correlations were found between BMI levels and CHDRPS in males (p < 0.001), females (p < 0.001), WNHs (p < 0.008), Hs (p < 0.001), WNH males (p < 0.024), H males (p < 0.004) and H females (p < 0.001). The mean knowledge of CHD questions of all subjects was 71.70 ± 7.92 out of 100. The mean knowledge of CHD was significantly higher for WNH males (p < 0.039) than BNH males. A significant inverse correlation (r = 0.392, p < 0.032) was found between the CHD knowledge and CHDRPS in WNH females. The researcher's findings indicate strong gender and ethnic differences in CHD risk factors among the college-age population. ^

Anti-diarrheal plants of central Anatolia: Do they inhibit diarrhea-causing bacteria?

Infectious diarrhea results in 2 to 5 million deaths worldwide per year, and treatments that are safe, effective, and readily available are under investigation. The field of medicinal ethnobotany focuses on plants that are used by different cultural groups for treating various diseases and evaluates these plants for efficacy and cytotoxicity. In the present study, ethnobotanical research was conducted with Central Anatolian villagers in Turkey. Folk concepts and etiologies surrounding diarrhea were analyzed, as were salient plant-based remedies for diarrhea. Reviewing the literature, 91 plant species were described as anti-diarrheal in all of Turkey. In Central Anatolia, villagers described 35 species. For continued research via bactericidal and bacteriostatic bioassays, 15 plants were selected. Methanolic and aqueous extracts of medicinally used plant parts were evaluated for inhibitory properties against 10 diarrhea-causing bacteria in the first bioassay, and later 21 bacteria in a second assay utilizing spectrophotometry. The cytotoxic properties were also evaluated in an Alamar Blue Assay using HepG-2, PC-3, and SkMEL-5 human cell lines. While several extracts showed bactericidal and bacteriostatic properties, the methanolic extract of R. canina galls inhibited the most bacteria at the lowest concentrations. They were not cytotoxic. Thus, R. canina methanolic gall extracts were selected for bio-assay guided fractionation. Antibacterial activity was maintained in the third fraction which was composed of almost pure ellagic acid. The bioassay was repeated with standard ellagic acid, and the polyphenol retained potency in inhibiting multiple bacterial strains. Several other extracts showed promise for safe, effective anti-bacterial remedies for diarrhea.