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.     

Association of Total and Central Adiposity Measures with Fasting Insulin in a Biracial Population of Young Adults with Normal Glucose Tolerance: the CARDIA Study

SIDNEY, STEPHEN, CORA E. LEWIS, JAMES O. HILL, CHARLES P. QUESENBERRY, JR, ELIZABETH R. STAMM, ANN SCHERZINGER, KIMBERLY TOLAN, AND BRUCE ETTINGER. Association of total and central adiposity measures with fasting insulin in a biracial population of young adults with normal glucose tolerance: the CARDIA study. Obes Res. Objective: To determine the association of computed tomography (CT)-measured visceral adipose tissue (AT) and other measures of adiposity with fasting insulin in a biracial (African American and Caucasian) study population of young adults. Research Methods and Procedures: The study population consisted of 251 young adults with normal glucose tolerance (NGT), ages 28–40 years, who were volunteers from the Birmingham, Alabama, and Oakland, California centers of the Coronary Artery Risk Development in Young Adults (CARDIA) study. Results: In regression models with total adiposity measures (body mass index or dual-energy X-ray absorptiometry-measured percent fat), visceral AT (measured as a cross-sectional area in cm²) was generally a stronger predictor of insulin than overall adiposity in all race/gender groups (partial correlation coefficients ranging from 0. 31 to 0. 47) except for black men, in whom the associations were nonsignificant. Partial correlation coefficients between waist circumference and insulin, controlling for percent fat, were nearly identical to those between visceral AT and insulin in women and in white men. Analyses performed on 2060 NGT CARDIA subjects who were not in this study of visceral AT showed significant correlations of waist circumference with insulin in all racelgender groups, including black men, and that black men in the visceral AT study group were significantly leaner than other black male CARDIA subjects. Discussion: We conclude that visceral AT was associated with fasting insulin in NGT participants in three of the four race/gender groups (black men excepted) and that waist circumference was a good surrogate for visceral AT in examining associations of central adiposity with fasting insulin.     

Borrelia burgdorferi binds fibronectin through a tandem beta-zipper, a common mechanism of fibronectin binding in staphylococci, streptococci, and spirochetes

BBK32 is a fibronectin-binding protein from the Lyme disease-causing spirochete Borrelia burgdorferi. In this study, we show that BBK32 shares sequence similarity with fibronectin module-binding motifs previously identified in proteins from Streptococcus pyogenes and Staphylococcus aureus. Nuclear magnetic resonance spectroscopy and isothermal titration calorimetry are used to confirm the binding sites of BBK32 peptides within the N-terminal domain of fibronectin and to measure the affinities of the interactions. Comparison of chemical shift perturbations in fibronectin F1 modules on binding of peptides from BBK32, FnBPA from S. aureus, and SfbI from S. pyogenes provides further evidence for a shared mechanism of binding. Despite the different locations of the bacterial attachment sites in BBK32 compared with SfbI from S. pyogenes and FnBPA from S. aureus, an antiparallel orientation is observed for binding of the N-terminal domain of fibronectin to each of the pathogens. Thus, these phylogenetically and morphologically distinct bacterial pathogens have similar mechanisms for binding to human fibronectin.     

Proteomic Studies of a Single CNS Synapse Type: The Parallel Fiber/Purkinje Cell Synapse

Precise neuronal networks underlie normal brain function and require distinct classes of synaptic connections. Although it has been shown that certain individual proteins can localize to different classes of synapses, the biochemical composition of specific synapse types is not known. Here, we have used a combination of genetically engineered mice, affinity purification, and mass spectrometry to profile proteins at parallel fiber/Purkinje cell synapses. We identify approximately 60 candidate postsynaptic proteins that can be classified into 11 functional categories. Proteins involved in phospholipid metabolism and signaling, such as the protein kinase MRCKγ, are major unrecognized components of this synapse type. We demonstrate that MRCKγ can modulate maturation of dendritic spines in cultured cortical neurons, and that it is localized specifically to parallel fiber/Purkinje cell synapses in vivo. Our data identify a novel synapse-specific signaling pathway, and provide an approach for detailed investigations of the biochemical complexity of central nervous system synapse types.     

Copper induced oxidation of serotonin: analysis of products and toxicity

Serotonin is a major neurotransmitter that controls many functions, ranging from mood and behaviour through to sleep and motor functions. The non-enzymatic oxidation of serotonin is of significant importance as some oxidation products are considered to be neurotoxic. An interaction between copper and serotonin has been suggested by symptoms observed in a number of neurodegenerative diseases such as Wilson's and Prion diseases. Using PC12 cells as a model of neuronal cells, we show that the interaction between copper and serotonin is toxic to undifferentiated cells. The toxicity is largely due to reactive oxygen species as cell death is significantly reduced in the presence of the antioxidant mannitol. Differentiation of the PC12 cells also confers resistance to the oxidative process. In vitro oxidation of serotonin by copper results in the eventual formation of a coloured pigment, thought to be a melanin-like polymeric species. Using spectroscopic methods we provide evidence for the formation of a single intermediate product. This dimeric intermediate was identified and characterized as 5,5'-dihydroxy-4,4'-bitryptamine. These results indicate that copper structurally alters serotonin and this process may play a role in copper related neurodegenerative diseases.     

Water-deficit induction of a tomato H1 histone requires abscisic acid

Many genes are induced by periods of water deficit, and a subset of these are dependent on elevated ABA content for expression. A number of drought-induced genes are not induced in leaves of the ABA-deficient mutant flacca from tomato (Lycopersicon esculentum) but are induced in detached, wilted wild-type leaves and ABA-treated leaves of both genotypes. The nucleotide sequence of the cDNA and corresponding genomic DNA fragment of one of these genes, his1-s (formerly called le20), encodes an amino acid sequence that is rich in Lys, Ala, and Ser. The predicted protein contains the tripartite structure of H1 histone and is similar to other H1 histones, especially in the globular domain. Since, his1-s is more closely related to a stress-induced gene from Lycopersicon pennellii than to another H1 histone in the tomato genome it is considered a stress-induced variant of H1 histone. his1-s mRNA accumulated in vegetative plants in response to other abiotic stress treatments, including application of polyethylene glycol, and salt. The mRNA preferentially accumulated in leaves as compared to roots. his1-s mRNA accumulation was controlled during development; the level was higher in developing seeds of mature green fruit than in detached wilted leaves. H1 histones have been implicated in the general repression of gene expression and in the regulation of specific genes. The rapid accumulation of his1-s mRNA during stress may indicate that this unique, stress-induced H1 histone is involved in controlling gene expression during plant stress.     

Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis

1. Download : Download high-res image (226KB)
2. Download : Download full-size image

     

Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe,Protective Vaccine Antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.