Multiple initial culture conditions enhance the establishment of cell lines from primary ovarian cancer specimens

Summary To increase the efficiency of stable cell line establishment from primary ovarian cancer specimens, we simultaneously initiated cultures under, multiple conditions, varying extracellular matrices and the inclusion of supplements (e.g., serum or serum albumin), while minimizing exposure to xenogeneic antigens (e.g., fetal calf serum). Primary cultures were initiated from 30 specimens; cell lines were established from 10 of these for a success rate of 33%. In some instances, multiple cell lines were established from the same specimen. Five lines were characterized extensively with respect to growth properties, antigen expression, and genomic alterations. Although these lines are all low-passage, marked heterogeneity was observed, even between lines derived from the same specimen. The culture approach outlined herein will facilitate generation of reagents useful for many aspects of ovarian cancer biology. Equal contribution.     

ABCG1-mediated generation of extracellular cholesterol microdomains

Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 μg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space.     

Increased incidence of vaginal septum in C57BL/6J mice since 1976

Decreased fertility was observed in a breeding colony of C57BL/6J mice. On examination, a dorsoventral vaginal septum was detected in many females. This defect was identified in 1976, with incidence of 4.0% in this strain. Our objective was to determine whether incidence of this condition has increased and whether this defect was associated with the observed infertility. We report incidence of 11.3%, nearly triple the original reported incidence. For comparison, incidence of vaginal septum in C57BL/6N females was determined and was found to be 1%. We performed a breeding study using normal and affected C57BL/6J females to evaluate fertility in affected females. Our data were consistent with those of the 1976 report; fertility was decreased in females with an intact vaginal septum. In 50% of affected females, the septum remained intact after breeding. The fertility for this subgroup of vaginal septum-retained females was 14.3%, compared with 85.7% in females whose septum ruptured and 75.0% in normal females (statistically significant, P = 0.02). On the basis of our results, we provide animal and financial loss data due to the defect. Lastly, we provide suggestions on how to minimize animal losses and be in accordance with the principles of the 3Rs (replacement, refinement, reduction).     

Modulation of Nrf2/ARE Pathway by Food Polyphenols: A Nutritional Neuroprotective Strategy for Cognitive and Neurodegenerative Disorders

In recent years, there has been a growing interest, supported by a large number of experimental and epidemiological studies, for the beneficial effects of some phenolic substances, contained in commonly used spices and herbs, in preventing various age-related pathologic conditions, ranging from cancer to neurodegenerative diseases. Although the exact mechanisms by which polyphenols promote these effects remain to be elucidated, several reports have shown their ability to stimulate a general xenobiotic response in the target cells, activating multiple defense genes. Data from our and other laboratories have previously demonstrated that curcumin, the yellow pigment of curry, strongly induces heme-oxygenase-1 (HO-1) expression and activity in different brain cells via the activation of heterodimers of NF-E2-related factors 2 (Nrf2)/antioxidant responsive element (ARE) pathway. Many studies clearly demonstrate that activation ofNrf2 target genes, and particularly HO-1, in astrocytes and neurons is strongly protective against inflammation, oxidative damage, and cell death. In the central nervous system, the HO system has been reported to be very active, and its modulation seems to play a crucial role in the pathogenesis of neurodegenerative disorders. Recent and unpublished data from our group revealed that low concentrations of epigallocatechin-3-gallate, the major green tea catechin, induces HO-1 by ARE/Nrf2 pathway in hippocampal neurons, and by this induction, it is able to protect neurons against different models of oxidative damages. Furthermore, we have demonstrated that other phenolics, such as caffeic acid phenethyl ester and ethyl ferulate, are also able to protect neurons via HO-1 induction. These studies identify a novel class of compounds that could be used for therapeutic purposes as preventive agents against cognitive decline.     

Bmp signaling is required for development of primary lens fiber cells

We have investigated the role of Bmp signaling in development of the mouse lens using three experimental strategies. First, we have shown that the Bmp ligand inhibitor noggin can suppress the differentiation of primary lens fiber cells in explant culture. Second, we have expressed a dominant-negative form of the type 1 Bmp family receptor Alk6 (Bmpr1b -- Mouse Genome Informatics) in the lens in transgenic mice and shown that an inhibition of primary fiber cell differentiation can be detected at E13.5. Interestingly, the observed inhibition of primary fiber cell development was asymmetrical and appeared only on the nasal side of the lens in the ventral half. Expression of the inhibitory form of Alk6 was driven either by the alpha A-cystallin promoter or the ectoderm enhancer from the Pax6 gene in two different transgenes. These expression units drive transgene expression in distinct patterns that overlap in the equatorial cells of the lens vesicle at E12.5. Despite the distinctions between the transgenes, they caused primary fiber cell differentiation defects that were essentially identical, which implied that the equatorial lens vesicle cells were responding to Bmp signals in permitting primary fiber cells to develop. Importantly, E12.5 equatorial lens vesicle cells showed cell-surface immunoreactivity for bone-morphogenetic protein receptor type 2 and nuclear immunoreactivity for the active, phosphorylated form of the Bmp responsive Smads. This indicated that these cells had the machinery for Bmp signaling and were responding to Bmp signals. We conclude that Bmp signaling is required for primary lens fiber cell differentiation and, given the asymmetry of the differentiation inhibition, that distinct differentiation stimuli may be active in different quadrants of the eye.     

Viewpoints on Factors for Successful Employment for Adults with Autism Spectrum Disorder

This article explores the key factors for successful employment from the viewpoints of adults with autism spectrum disorder (ASD) and employers. Two groups of individuals participated in this study, 40 adults with ASD and 35 employers. Q method was used to understand and contrast the viewpoints of the two groups. Data were analysed using by-person varimax rotation factor analysis. Results showed that although both groups appear committed to the employment process, the difference in their understanding regarding the type of workplace support required, job expectations and productivity requirements continues to hinder successful employment. These results highlight the need to facilitate communication between employees and employers to ensure a clear understanding of the needs of both groups are met. The use of an ASD-specific workplace tool may assist in facilitating the necessary communication between these two groups.     

The cAMP signaling pathway has opposing effects on Rac and Rho in B16F10 cells: implications for dendrite formation in melanocytic cells

A hallmark of melanocytic cells is their ability to form dendrites in response to growth factors and to ultraviolet irradiation. It is known that the cyclic adenosine monophosphate (cAMP) second messenger pathway stimulates melanocyte dendrite formation because agents that increase cAMP such as forskolin and dibutyrl cAMP induce dendrite formation in normal human and murine melanocytes and melanoma cell lines. The Rho family of guanosine triphosphate (GTP)-binding proteins regulates cytoskeletal reorganization in all cells tested and Rac and Rho have both been shown to regulate melanocyte dendrite formation. In this report, we analyzed the effect of cAMP on the activation of Rac and Rho and show that elevation of cAMP stimulates Rac and inhibits Rho in B16F10 cells. The Rho GTP-binding proteins have also been shown to either cross-activate or inhibit each other and in this report we show that Rac activates Rho in B16F10 cells. Microinjection of C3 botulinum exoenzyme toxin, an agent that specifically inactivates Rho or microinjection of constitutively active mutant Rac protein-induced dendricity in human melanocytes and in B16F10 and B16F1 murine melanoma cell lines. We conclude that cAMP-mediated dendrite formation in melanocytic cells is mediated through upregulation of Rac activity and downregulation of Rho activity.