Proteomics analysis of chinese hamster ovary cells undergoing apoptosis during prolonged cultivation

The degradation of environmental conditions, such as nutrient depletion and accumulation of toxic waste products over time, often lead to premature apoptotic cell death in mammalian cell cultures and suboptimal protein yield. Although apoptosis has been extensively researched, the changes in the whole cell proteome during prolonged cultivation, where apoptosis is a major mode of cell death, have not been examined. To our knowledge, the work presented here is the first whole cell proteome analysis of non-induced apoptosis in mammalian cells. Flow cytometry analyses of various activated caspases demonstrated the onset of apoptosis in Chinese hamster ovary cells during prolonged cultivation was primarily through the intrinsic pathway. Differential in gel electrophoresis proteomic study comparing protein samples collected during cultivation resulted in the identification of 40 differentially expressed proteins, including four cytoskeletal proteins, ten chaperone and folding proteins, seven metabolic enzymes and seven other proteins of varied functions. The induction of seven ER chaperones and foldases is a solid indication of the onset of the unfolded protein response, which is triggered by cellular and ER stresses, many of which occur during prolonged batch cultures. In addition, the upregulation of six glycolytic enzymes and another metabolic protein emphasizes that a change in the energy metabolism likely occurred as culture conditions degraded and apoptosis advanced. By identifying the intracellular changes during cultivation, this study provides a foundation for optimizing cell line-specific cultivation processes, prolonging longevity and maximizing protein production.     

Maintenance of HIV-specific CD4+ T cell help distinguishes HIV-2 from HIV-1 infection

Unlike HIV-1-infected people, most HIV-2-infected subjects maintain a healthy CD4+ T cell count and a strong HIV-specific CD4+ T cell response. To define the cellular immunological correlates of good prognosis in HIV-2 infection, we conducted a cross-sectional study of HIV Gag-specific T cell function in HIV-1- and HIV-2-infected Gambians. Using cytokine flow cytometry and lymphoproliferation assays, we show that HIV-specific CD4+ T cells from HIV-2-infected individuals maintained proliferative capacity, were not terminally differentiated (CD57-), and more frequently produced IFN-gamma or IL-2 than CD4+ T cells from HIV-1-infected donors. Polyfunctional (IFN-gamma+/IL-2+) HIV-specific CD4+ T cells were found exclusively in HIV-2+ donors. The disparity in CD4+ T cell responses between asymptomatic HIV-1- and HIV-2-infected subjects was not associated with differences in the proliferative capacity of HIV-specific CD8+ T cells. This study demonstrates that HIV-2-infected donors have a well-preserved and functionally heterogeneous HIV-specific memory CD4+ T cell response that is associated with delayed disease progression in the majority of infected people.     

Selection of Fruit by Gibbons (Hylobates muelleri × agilis) in the Rain Forests of Central Borneo

Gibbons (Hylobates spp.) are among the main frugivorous primates in Southeast Asia, yet little is known about the criteria by which they select fruit for consumption. We studied two gibbon groups for 14 mo in the lowland dipterocarp forests of Central Borneo to determine their selectivity for different fruit species and traits. Ideal gibbon fruit were yellow, large, with a juicy-soft pulp, thin skin and available in large crops. Gibbons ultimately sought seedless fruit, but when seeds were present they selected fruit with a single, well-protected seed. Given that few fruit exhibited all the desired traits, we also carried out a multiple regression using the selection ratios of the various fruit species and their associated fruit traits to determine which traits ultimately determined gibbon choice. The analysis was stratified to account for differences in fruit availability. Selection was strongest when fruit were abundant in the forest and was based on seed width (<21 mm), color (yellow-orange), and fruit weight (1–5 g). No selection is apparent when food abundance was intermediate, but when fruit were scarce they preferentially ate larger fruit (6–30 g).     

High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

Background

Novel tuberculosis (TB) vaccines recently tested in humans have been designed to boost immunity induced by the current vaccine, Mycobacterium bovis Bacille Calmette-Guérin (BCG). Because BCG vaccination is used extensively in infants, this population group is likely to be the first in which efficacy trials of new vaccines will be conducted. However, our understanding of the complexity of immunity to BCG in infants is inadequate, making interpretation of vaccine-induced immune responses difficult.

Methods

To better understand BCG-induced immunity, we performed gene expression profiling in five 10-week old infants routinely vaccinated with BCG at birth. RNA was extracted from 12 hour BCG-stimulated or purified protein derivative of tuberculin (PPD)-stimulated PBMC, isolated from neonatal blood collected 10 weeks after vaccination. RNA was hybridised to the Sentrix^® HumanRef-8 Expression BeadChip (Illumina) to measure expression of >16,000 genes.

Results

We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation. The peroxisome proliferator-activated receptor (PPAR) signaling pathway, including PPAR-γ, involved in activation of the alternative, anti-inflammatory macrophage response was down-regulated following stimulation with both antigens. In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted. Further analysis revealed a decrease in the expression of cell adhesion molecules (CAMs), including integrin alpha M (ITGAM), which is known to be important for entry of mycobacteria into the macrophage. Interestingly, more leukocyte genes were down-regulated than up-regulated.

Conclusion

Our results suggest that a combination of suppressed and up-regulated genes may be key in determining development of protective immunity to TB induced by vaccination with BCG.     

Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans

In animals, effective immune responses against malignancies and against several infectious pathogens, including malaria, are mediated by T cells. Here we show that a heterologous prime-boost vaccination regime of DNA either intramuscularly or epidermally, followed by intradermal recombinant modified vaccinia virus Ankara (MVA), induces high frequencies of interferon (IFN)-gamma-secreting, antigen-specific T-cell responses in humans to a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP). These responses are five- to tenfold higher than the T-cell responses induced by the DNA vaccine or recombinant MVA vaccine alone, and produce partial protection manifest as delayed parasitemia after sporozoite challenge with a different strain of Plasmodium falciparum. Such heterologous prime-boost immunization approaches may provide a basis for preventative and therapeutic vaccination in humans.     

Metabolic reconstruction and analysis for parasite genomes

With the completion of sequencing projects for several parasite genomes, efforts are ongoing to make sense of this mass of information in terms of the gene products encoded and their interactions in the growth, development and survival of parasites. The emerging science of systems biology aims to explain the complex relationship between genotype and phenotype by using network models. One area in which this approach has been particularly successful is in the modeling of metabolism. With an accurate picture of the set of metabolic reactions encoded in a genome, it is now possible to identify enzymes or transporters that might be viable targets for new drugs. Because these predictions greatly depend on the quality and completeness of the genome annotation, there are substantial efforts in the scientific community to increase the numbers of metabolic enzymes identified. In this review, we discuss the opportunities for using metabolic reconstruction and analysis tools in parasitology research, and their applications to protozoan parasites.     

Glucagon activation of the thiol:protein disulfide oxidoreductase in isolated, rat, hepatic microsomes

The hepatic, microsomal, thiol:protein disulfide oxidoreductase catalyzes the glutathione (GSH) reduction of protein disulfides to sulfhydryl groups. In the presence of physiological concentrations of glucagon this activity increased from 2.3 to 6.4 fold in isolated microsomes. The stimulation had a P50 for glucagon of 7.8 X 10(-10) M which was only observed at microsomal protein concentrations of less than 100 micrograms/ml and in the presence of a GSH reducing system. This latter observation suggests that the stimulation may be inhibited by the presence of oxidized glutathione. These data support the hypothesis that glucagon may act in part by stimulating the reduction of protein disulfides by the thiol:protein disulfide oxidoreductase.