Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.     

Acid Ceramidase Promotes Nuclear Export of PTEN through Sphingosine 1-Phosphate Mediated Akt Signaling

The tumor suppressor PTEN is now understood to regulate cellular processes at the cytoplasmic membrane, where it classically regulates PI3K signaling, as well as in the nucleus where multiple roles in controlling cell cycle and genome stability have been elucidated. Mechanisms that dictate nuclear import and, less extensively, nuclear export of PTEN have been described, however the relevance of these processes in disease states, particularly cancer, remain largely unknown. We investigated the impact of acid ceramidase on the nuclear-cytoplasmic trafficking of PTEN. Immunohistochemical analysis of a human prostate tissue microarray revealed that nuclear PTEN was lost in patients whose tumors had elevated acid ceramidase. We found that acid ceramidase promotes a reduction in nuclear PTEN that is dependent upon sphingosine 1-phosphate-mediated activation of Akt. We were further able to show that sphingosine 1-phosphate promotes formation of a complex between Crm1 and PTEN, and that leptomycin B prevents acid ceramidase and sphingosine 1-phosphate mediated loss of nuclear PTEN, suggesting an active exportin-mediated event. To investigate whether the tumor promoting aspects of acid ceramidase in prostate cancer depend upon its ability to export PTEN from the nucleus, we used enforced nuclear expression of PTEN to study docetaxel-induced apoptosis and cell killing, proliferation, and xenoengraftment. Interestingly, while acid ceramidase was able to protect cells expressing wild type PTEN from docetaxel, promote proliferation and xenoengraftment, acid ceramidase had no impact in cells expressing PTEN-NLS. These findings suggest that acid ceramidase, through sphingosine 1-phosphate, promotes nuclear export of PTEN as a means of promoting tumor formation, cell proliferation, and resistance to therapy.     

The in vivo dose rate effect of chronic gamma radiation in mice: translocation and micronucleus analyses

The in vivo effects of chronic, ultra low dose rates of gamma radiation in mice were evaluated using fluorescence in situ hybridization and the in vivo micronucleus test. SWR×C57BL/6 mice were divided into nine exposure groups and continuously exposed to 0.5, 2.0 or 4.0 cGy ¹³⁷Cs per day for 30, 60 or 90 days; unexposed control mice were also included. Following exposure, blood samples were taken from each animal and the frequencies of micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE) were determined using flow cytometry. Peripheral blood lymphocytes were cultured and analyzed by chromosome painting to determine translocation frequencies. A significant dose rate response was seen in translocations and both MPCE and MNCE. Comparisons were made between the three chronic dose rates and it was determined that there was no significant difference among translocation frequencies for each rate. However, a significant difference was found between the chronic exposures reported here and the fractionated daily exposures reported previously. Dose rate reduction effects, ranging from 3 at low doses to 14 at high doses, were found for chronic versus acute exposures. The possibility of gender effects was investigated in both micronucleus and translocation data. No gender effect was found in translocation induction, but a slight effect was suggested in micronucleus induction.     

Muscle palmitate uptake and binding are saturable and inhibited by antibodies to FABPPM

Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABP_PM) may be a component of this system. To test the hypothesis that FABP_PM is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABP_PM. Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated V_max values of 10.5 ± 1.2 pmol/mg protein/15 sec and 45.6 ± 2.9 nmol/mg protein/15 min and K_m values of 12.8 ± 3.8 and 18.4 ± 1.8 nmol/L, respectively. The V_max values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABP_PM. Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABP_PM. Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABP_PM is a component of this process in muscle.     

Transcontinental Phylogeography of the Daphnia pulex Species Complex

Daphnia pulex is quickly becoming an attractive model species in the field of ecological genomics due to the recent release of its complete genome sequence, a wide variety of new genomic resources, and a rich history of ecological data. Sequences of the mitochondrial NADH dehydrogenase subunit 5 and cytochrome c oxidase subunit 1 genes were used to assess the global phylogeography of this species, and to further elucidate its phylogenetic relationship to other members of the Daphnia pulex species complex. Using both newly acquired and previously published data, we analyzed 398 individuals from collections spanning five continents. Eleven strongly supported lineages were found within the D. pulex complex, and one lineage in particular, panarctic D. pulex, has very little phylogeographical structure and a near worldwide distribution. Mismatch distribution, haplotype network, and population genetic analyses are compatible with a North American origin for this lineage and subsequent spatial expansion in the Late Pleistocene. In addition, our analyses suggest that dispersal between North and South America of this and other species in the D. pulex complex has occurred multiple times, and is predominantly from north to south. Our results provide additional support for the evolutionary relationships of the eleven main mitochondrial lineages of the D. pulex complex. We found that the well-studied panarctic D. pulex is present on every continent except Australia and Antarctica. Despite being geographically very widespread, there is a lack of strong regionalism in the mitochondrial genomes of panarctic D. pulex – a pattern that differs from that of most studied cladocerans. Moreover, our analyses suggest recent expansion of the panarctic D. pulex lineage, with some continents sharing haplotypes. The hypothesis that hybrid asexuality has contributed to the recent and unusual geographic success of the panarctic D. pulex lineage warrants further study.     

Antigenic analysis of a major neutralization site of herpes simplex virus glycoprotein D, using deletion mutants and monoclonal antibody-resistant mutants.

Herpes simplex virus glycoprotein D is a component of the virion envelope and appears to be involved in attachment, penetration, and cell fusion. Monoclonal antibodies against this protein can be arranged in groups on the basis of a number of biological and biochemical properties. Group I antibodies are type common, have high complement-independent neutralization titers, and recognize discontinuous (conformational) epitopes; they are currently being used in several laboratories to study the functions of glycoprotein D. We have used a panel of neutralization-resistant mutants to examine the relationships between these antibodies in detail. We found that they can be divided into two subgroups, Ia and Ib, such that mutations selected with Ia antibodies have little or no effect on binding and neutralization by Ib antibodies and vice versa. In addition, Ia antibodies are able to bind deletion and truncation mutants of glycoprotein D that Ib antibodies do not recognize, suggesting that their epitopes are physically distinct. However, with one exception, Ia and Ib antibodies block each other strongly in binding assays with purified glycoprotein D, whereas antibodies from other groups have no effect. We have therefore defined the sum of the Ia and Ib epitopes as antigenic site 1.     

Antisense knockdown of the beta1 integrin subunit in the chicken embryo results in abnormal neural crest cell development

Neural crest cells escape the neural tube by undergoing an epithelial to mesenchymal transition (EMT). This is followed by extensive migration along specific pathways that are lined with extracellular matrix (ECM). In this study, we have examined the roles of matrix receptors containing beta1 integrin subunits in neural crest cell morphogenesis using antisense morpholino oligos electroporated in ovo into avian neural crest cell precursors. Our results show that reduced levels of expression of beta1 integrin subunits in the dorsal neural tube results in an abnormal epithelial to mesenchymal transition. In approximately half of the experimental embryos, however, some neural crest cells filled with beta1 antisense are able to escape the neural tube and migrate ventrally, indicating that grossly normal migration of trunk neural crest cells can take place after beta1 integrin expression is reduced. This study shows the potential of this novel method for investigating the roles of genes that are required for the survival of early mouse embryos in later development events.