Can soy phytoestrogens decrease DNA methylation in BRCA1 and BRCA2 oncosuppressor genes in breast cancer?

Although soy phytoestrogens have been postulated to exert a protective effect against breast cancer, the attendant mechanisms, in particular epigenetics underpinnings, have remained elusive. We investigated the putative effects on DNA methylation by two naturally occurring isoflavones, genistein and daidzein, in a study of the BRCA1 and BRCA2 oncosuppressor genes in breast cancer cell lines (MCF-7, MDA-MB 231, and MCF10a). A demethylant agent, the 5-azacytidine, and a methylant, the budesonide, were used as treatment controls. DNA methylation of BRCA1 and BRCA2 was investigated with methylated DNA immunoprecipitation coupled with PCR. In parallel, protein expression was determined by Western blot, immunohistochemistry, and confocal microscopy. Our results suggest that treatment with 18.5?μM Genistein or 78.5?μM Daidzein might reverse DNA hypermethylation and restore the expression of the oncosuppressor genes BRCA1 and BRCA2. 5-Azacitydine also enhanced the reexpression of these genes while budesonide had an opposite effect. To the best of our knowledge, these observations, while requiring replication, provide new evidence on potential epigenetic mechanisms by which genistein and daidzein might contribute to regulation of the BRCA1 and BRCA2. Future studies are warranted on whether the demethylating effect of genistein and daidzein is global or focused on select candidate genes.     

Anoctamin 1 dysregulation alters bronchial epithelial repair in cystic fibrosis

Cystic fibrosis (CF) airway epithelium is constantly subjected to injury events due to chronic infection and inflammation. Moreover, abnormalities in CF airway epithelium repair have been described and contribute to the lung function decline seen in CF patients. In the last past years, it has been proposed that anoctamin 1 (ANO1), a Ca^2 +-activated Cl^? channel, might offset the CFTR deficiency but this protein has not been characterized in CF airways. Interestingly, recent evidence indicates a role for ANO1 in cell proliferation and tumor growth. Our aims were to study non-CF and CF bronchial epithelial repair and to determine whether ANO1 is involved in airway epithelial repair. Here, we showed, with human bronchial epithelial cell lines and primary cells, that both cell proliferation and migration during epithelial repair are delayed in CF compared to non-CF cells. We then demonstrated that ANO1 Cl^? channel activity was significantly decreased in CF versus non-CF cells. To explain this decreased Cl^? channel activity in CF context, we compared ANO1 expression in non-CF vs. CF bronchial epithelial cell lines and primary cells, in lung explants from wild-type vs. F508del mice and non-CF vs. CF patients. In all these models, ANO1 expression was markedly lower in CF compared to non-CF. Finally, we established that ANO1 inhibition or overexpression was associated respectively with decreases and increases in cell proliferation and migration. In summary, our study demonstrates involvement of ANO1 decreased activity and expression in abnormal CF airway epithelial repair and suggests that ANO1 correction may improve this process.     

Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension

Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x_L and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x_L over-expressing subline (hVMbcl-x_L) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-x_L cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 ± 0.8% to 17.2 ± 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-x_L-over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-x_L cell cultures compared with control. We conclude that Bcl-x_L and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells.     

Cellular Functions and X-ray Structure of Anthrolysin O, a Cholesterol-dependent Cytolysin Secreted by Bacillus anthracis

Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin (CDC) secreted by Bacillus anthracis, the etiologic agent for anthrax. Growing evidence suggests the involvement of ALO in anthrax pathogenesis. Here, we show that the apical application of ALO decreases the barrier function of human polarized epithelial cells as well as increases intracellular calcium and the internalization of the tight junction protein occludin. Using pharmacological agents, we also found that barrier function disruption requires increased intracellular calcium and protein degradation. We also report a crystal structure of the soluble state of ALO. Based on our analytical ultracentrifugation and light scattering studies, ALO exists as a monomer. Our ALO structure provides the molecular basis as to how ALO is locked in a monomeric state, in contrast to other CDCs that undergo antiparallel dimerization or higher order oligomerization in solution. ALO has four domains and is globally similar to perfringolysin O (PFO) and intermedilysin (ILY), yet the highly conserved undecapeptide region in domain 4 (D4) adopts a completely different conformation in all three CDCs. Consistent with the differences within D4 and at the D2-D4 interface, we found that ALO D4 plays a key role in affecting the barrier function of C2BBE cells, whereas PFO domain 4 cannot substitute for this role. Novel structural elements and unique cellular functions of ALO revealed by our studies provide new insight into the molecular basis for the diverse nature of the CDC family.Cholesterol-dependent cytolysins (CDCs)⁴ are a family of pore-forming toxins from many organisms, including but not limited to the genera Archanobacterium, Bacillus, Clostridium, Listeria, and Streptococcus. Recently, work in vertebrates has revealed that CDCs and membrane attack complex/perforin superfamily domain-containing proteins share a similar fold, suggesting that vertebrates use a similar mechanism for defense against infection (1, 2). A common feature of the CDC family is the requirement of cholesterol in the membrane to form pores (3). In addition to cholesterol, certain members of the family also require a cellular receptor, such as CD59 for the toxin ILY from Streptococcus intermedius (4). The specific mechanism by which CDCs form pores is not completely resolved; however, what is generally known is that ring-shaped oligomerization at the cellular membrane is followed by large conformational changes in each unit of the oligomer, resulting in the insertion of a β-barrel into the cellular membrane (5). Pore formation results in a variety of downstream signaling effects, including but not limited to the influx of Ca²⁺ into the cell (6).A good deal is known about structures of the prepore conformation of CDCs. The crystal structures of prepore PFO, from Clostridium perfringens, and ILY have previously been elucidated (7, 8). Each structure shows a characteristic four-domain architecture, in which domain 4 (D4) is involved in membrane recognition, domain 3 (D3) is involved in β-sheet insertion, and domain 2 (D2) is the hinge region that undergoes a large conformational change (9-11). Nevertheless, despite the similarities, structural differences in D4 orientation and the conformation of a highly conserved segment named the undecapeptide region confer functional differences to PFO and ILY (8). Noting these differences, we decided to explore the structure and function of another member of the CDC family, anthrolysin O (ALO).ALO is secreted by Bacillus anthracis, the etiologic agent for anthrax. ALO is chromosomally encoded by a gene whose regulation is poorly understood, and it is highly homologous to other members of the CDC family (12). ALO has been shown to have hemolytic and cytolytic activity (13, 14). Although clinical studies have shown that B. anthracis is weakly hemolytic (15), anthrax bacteria do produce biologically relevant amounts of hemolytic ALO, although the levels of expression are under complex regulation and are dependent on the culture media and growth conditions (12, 13, 16). At lower concentrations, ALO can disrupt cell signaling (13, 14). Search for a cellular receptor of ALO has lead to the conclusion that it is a TLR4 agonist (17). However, it is not known that ALO binds to TLR4 directly and, if so, whether ALO also binds other cellular receptors.In addition to ALO, B. anthracis secrete ∼400 proteins, termed the anthrax secretome (18). Of those, two exotoxins, edema toxin (ET) and lethal toxin (LT) have been characterized in greatest detail. ET raises intracellular cAMP to pathologic levels, whereas LT impairs mitogenic and stress responses by inactivating mitogen-activating protein kinase kinase (19, 20). The complex interplay between these two toxins on various aspects of host cellular functions have been demonstrated (20-25). ALO could also work in conjunction with other anthrax virulence factors to modulate their cellular toxicity. For example, ALO and LF together induce macrophage apoptosis, whereas ALO and PLC play a redundant role in a murine inhalation anthrax model (17, 26). Interplay among anthrax secreted factors on cells relevant to anthrax infection is just beginning to be understood. This network of interactions is vital to the molecular basis of how anthrax bacteria interact with the hosts during anthrax infection.Anthrax infection initiates when B. anthracis spores enter the host through one of three routes: cutaneous, inhalational, or gastrointestinal (GI) (27, 28). All three routes of infection can lead to systemic infection and are ultimately lethal. Different from inhalational anthrax, spores are ingested and germinate on or within the epithelium of the GI tract in GI anthrax (29). This is primarily based on pathological observations that primary lesions of the GI tract are found in GI anthrax, whereas no primary lesions of the lung are found in inhalational anthrax (29). Inhalational anthrax is a disease of choice for biological weapons because of its high infectivity and mortality (30). The initiation of GI anthrax requires much higher doses of spores than inhalational anthrax, and the molecular basis for the initiation of GI anthrax remains elusive (31).Since the primary function of GI epithelia is to control the flux of material into the body, disruption of this barrier can lead to movement of bacteria into the surrounding tissue (32). The barrier is produced by a matrix of transmembrane and membrane-associated proteins. These cell to cell contacts, or tight junctions, are sometimes altered during bacterial infection to specifically disrupt the barrier function of epithelial cells. Using a functional model for the gut epithelium, human gut epithelial Caco-2 brush border expressor (C2BBE) cells, we report that ALO decreases the barrier function of C2BBE cells through disruption of tight junctions. We also show that ALO disruption of barrier function is dependent on epithelial cell polarity. We also present the crystal structure of the soluble state of ALO and compare it with the known structures of other CDCs. In addition, we show that ALO exists primarily as a monomer, in contrast to its closely related homologue PFO, which exists as a dimer. Finally, we used domain swapping to examine the structural components that confer specificity of ALO to gut epithelial cells.     

Neuroticism, acculturation and the cortisol awakening response in Mexican American adults

Neuroticism is associated with greater susceptibility to the adverse effects of stress and greater exposure to the stressors associated with acculturation in U.S. born Mexican Americans. Neuroticism and acculturation have been associated with injury to crucial stress response systems and are known risk factors for certain mood and anxiety disorders. The purpose of the current study was to examine the effects of neuroticism, and acculturation on the cortisol awakening response (CAR) in healthy Mexican-American adults. Salivary cortisol samples were collected at awakening and 30, 45, and 60 min thereafter, on two consecutive weekdays from 59 healthy Mexican American adult males (26) and females (33), ages 18 to 38 years. Participants were assessed for level of neuroticism and acculturation. Data were analyzed using a mixed effects regression model with repeated measures at four time points. Results showed a significant Neuroticism×Acculturation×Time interaction. The CAR was virtually eliminated in highly acculturated Mexican Americans with greater Anglo orientation and high neuroticism compared with less acculturated Mexican Americans with greater Mexican orientation and lower neuroticism. Findings suggest that some Mexican Americans with high levels of neuroticism may be particularly susceptible to certain challenges and stressors associated with acculturation leading over time to the development of allostatic load, desensitization of the Hypothalamic CRF system and attenuation of the CAR.     

Differential gene expression from microarray analysis distinguishes woven and lamellar bone formation in the rat ulna following mechanical loading

Formation of woven and lamellar bone in the adult skeleton can be induced through mechanical loading. Although much is known about the morphological appearance and structural properties of the newly formed bone, the molecular responses to loading are still not well understood. The objective of our study was to use a microarray to distinguish the molecular responses between woven and lamellar bone formation induced through mechanical loading. Rat forelimb loading was completed in a single bout to induce the formation of woven bone (WBF loading) or lamellar bone (LBF loading). A set of normal (non-loaded) rats were used as controls. Microarrays were performed at three timepoints after loading: 1 hr, 1 day and 3 days. Confirmation of microarray results was done for a select group of genes using quantitative real-time PCR (qRT-PCR). The micorarray identified numerous genes and pathways that were differentially regulated for woven, but not lamellar bone formation. Few changes in gene expression were evident comparing lamellar bone formation to normal controls. A total of 395 genes were differentially expressed between formation of woven and lamellar bone 1 hr after loading, while 5883 and 5974 genes were differentially expressed on days 1 and 3, respectively. Results suggest that not only are the levels of expression different for each type of bone formation, but that distinct pathways are activated only for woven bone formation. A strong early inflammatory response preceded an increase in angiogenic and osteogenic gene expression for woven bone formation. Furthermore, at later timepoints there was evidence of bone resorption after WBF loading. In summary, the vast coverage of the microarray offers a comprehensive characterization of the early differences in expression between woven and lamellar bone formation.