Characteristics of the microsporidian Enterocytozoon bieneusi: a consequence of its development within short-living enterocytes.

Ultrastructural studies were done on developmental stages of Enterocytozoon bieneusi obtained from HIV seropositive patients suffering from diarrhea. The presence of elaborate multilamellar structures suggest that they give rise to various membrane systems needed for rapid production of disseminating stages.     

How inaccuracies in protein structure models affect estimates of protein-ligand interactions: computational analysis of HIV-I protease inhibitor binding

The influence of possible inaccuracies that can arise during homology modeling of protein structures used for ligand binding studies were investigated with the molecular mechanics generalized Born surface area (MM-GBSA) method. For this, a family of well-characterized HIV-I protease-inhibitor complexes was used. Validation of MM-GBSA led to a correlation coefficient ranging from 0.72 to 0.93 between calculated and experimental binding free energies DeltaG. All calculated DeltaG values were based on molecular dynamics simulations with explicit solvent. Errors introduced into the protein structure through misplacement of side-chains during rotamer modeling led to a correlation coefficient between DeltaG(calc) and DeltaG(exp) of 0.75 compared with 0.90 for the correctly placed side chains. This is in contrast to homology models for members of the retroviral protease family with template structures ranging in sequence identity between 32% and 51%. For these protein models, the correlation coefficients vary between 0.84 and 0.87, which is considerably closer to the original protein (0.90). It is concluded that HIV-I low sequence identity with the template structure still allows creating sufficiently reliable homology models to be used for ligand-binding studies, although placement of the rotamers is a critical step during the modeling.     

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, CK8, CK18, EGFR, MUC1, and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥ 40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.Key words: BRCA1, methylation, epigenetics, triple negative, breast cancer, retionblastoma tumor suppressor gene, pRb, p16     

Endothelial induced EMT in breast epithelial cells with stem cell properties

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.     

Expression and Functional Role of Sprouty-2 in Breast Morphogenesis

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.     

The Dsl1 tethering complex actively participates in soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) complex assembly at the endoplasmic reticulum in Saccharomyces cerevisiae

Intracellular transport is largely dependent on vesicles that bud off from one compartment and fuse with the target compartment. The first contact of an incoming vesicle with the target membrane is mediated by tethering factors. The tethering factor responsible for recruiting Golgi-derived vesicles to the ER is the Dsl1 tethering complex, which is comprised of the essential proteins Dsl1p, Dsl3p, and Tip20p. We investigated the role of the Tip20p subunit at the ER by analyzing two mutants, tip20-5 and tip20-8. Both mutants contained multiple mutations that were scattered throughout the TIP20 sequence. Individual mutations could not reproduce the temperature-sensitive phenotype of tip20-5 and tip20-8, indicating that the overall structure of Tip20p might be altered in the mutants. Using molecular dynamics simulations comparing Tip20p and Tip20-8p revealed that some regions, particularly the N-terminal domain and parts of the stalk region, were more flexible in the mutant protein, consistent with its increased susceptibility to proteolysis. Both Tip20-5p and Tip20-8p mutants prevented proper ER trans-SNARE complex assembly in vitro. Moreover, Tip20p mutant proteins disturbed the interaction between Dsl1p and the coatomer coat complex, indicating that the Dsl1p-coatomer interaction could be stabilized or regulated by Tip20p. We provide evidence for a direct role of the Dsl1 complex, in particular Tip20p, in the formation and stabilization of ER SNARE complexes.     

Docosahexaenoic acid in red blood cells of women of reproductive age is positively associated with oral contraceptive use and physical activity

Optimal intake of the long-chain n-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) and proper balance between intake of n-6 PUFA and n-3 PUFA are important for human health. Considerable evidence exists to show that DHA has a marked benefit during pregnancy. Lifestyle factors can affect the biosynthesis of DHA from dietary precursors, incorporation into membranes and degradation. The purpose of this study was to investigate the PUFA composition of red blood cells (RBCs) from women (n=40) in reproductive age, and how it is affected by diet and other lifestyle factors. Of all the lifestyle factors tested oral contraceptive use and physical activity were the ones correlated with DHA in RBCs, after adjustment for DHA intake. The findings indicate that oral contraceptive use and physical activity have a positive impact on the DHA status, as assessed by RBC level, of women in reproductive age.