Differential protein expression following low temperature culture of suspension CHO-K1 cells

<p>Abstract</p> <p>Background</p> <p>To ensure maximal productivity of recombinant proteins (rP) during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37°C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood.</p> <p>Results</p> <p>In this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37°C to 31°C in comparison to standard batch culture maintained at 37°C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry (MS). There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC), cap-independent translation (EIF4A), apoptosis (importin-α), the cytoskeleton (vimentin) and glycoprotein quality control (alpha glucosidase 2).</p> <p>Conclusion</p> <p>These results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. Regulation of these identified proteins and pathways could be useful for future approaches to engineer CHO cells for improved recombinant protein production.</p

Body mass index, prudent diet score and social class across three generations: evidence from the Hertfordshire Intergenerational Study.

BACKGROUND: Studies describing body mass index (BMI) and prudent diet score have reported that they are associated between parents and children. The Hertfordshire Intergenerational Study, which contains BMI, diet and social class information across three generations, provides an opportunity to consider the influence of grandparental and parental BMI and prudent diet score across multiple generations, and the influence of grandparental and parental social class on child BMI. METHODS: Linear regressions examining the tracking of adult BMI and prudent diet score across three generations (grandparent (F0), parent (F1) and child (F2)) were run from parent to child and from grandparent to grandchild. Linear mixed models investigated the influence of F0 and F1 BMI or prudent diet score on F2 BMI and prudent diet score. Linear regressions were run to determine whether social class and prudent diet score of parents and grandparents influenced the BMI of children and grandchildren. RESULTS: BMI was significantly associated across each generational pair and from F0 to F1 in multilevel models. Prudent diet score was significantly positively associated between grandparents and grandchildren. Lower grandparental and parental social class had a significantly positive association with F2 BMI (F0 low social class: b=1.188 kg/m2, 95% CI 0.060 to 2.315, p=0.039; F1 middle social class: b=2.477 kg/m2, 95% CI 0.726 to 4.227, p=0.006). CONCLUSION: Adult BMI tracks across generations of the Hertfordshire Intergenerational Study, and child BMI is associated with parental and grandparental social class. The results presented here add to literature supporting behavioural and social factors in the transmission of BMI across generations

Osteosarcopenia:Where Osteoporosis and Sarcopenia Collide

peer reviewedThe coexistence of osteoporosis and sarcopenia has been recently considered in some groups as a syndrome termed 'osteosarcopenia'. Osteoporosis describes low bone mass and deterioration of the micro-architecture of the bone, whereas sarcopenia is the loss of muscle mass, strength and function. With an ageing population the prevalence of both conditions is likely to increase substantially over the coming decades and is associated with significant personal and societal burden. The sequelae for an individual suffering from both conditions together include a greater risk of falls, fractures, institutionalization and mortality. The aetiology of 'osteosarcopenia' is multifactorial with several factors linking muscle and bone function, including genetics, age, inflammation and obesity. Several biochemical pathways have been identified that are facilitating the development of several promising therapeutic agents, which target both muscle and bone. In the current review we outline the epidemiology, pathogenesis and clinical consequences of 'osteosarcopenia' and explore current and potential future management strategies

Purification and Identification of a 7.6-kDa Protein in Media Conditioned by Superinvasive Cancer Cells

Background: Selection of the human drug sensitive and invasive cell line (MDA-MB-435S-F) with the chemotherapeutic agent paclitaxel, resulted in the development of drug resistant cell lines displaying enhanced invasion-related characteristics. Materials and Methods: Serum-free conditioned media from the human cancer drug-sensitive and invasive cell line (MDA-MB-435S-F) and its paclitaxel-resistant superinvasive variant (MDA-MB-435S-F/Taxol10p4pSI) were analyzed using Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Results: A differentially expressed protein was observed at 7.6 kDa, which was 4-fold upregulated in MDA-MB-435S-F/Taxol10p4pSI. The differentially expressed protein was identified using matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS), as a fragment of bovine transferrin. The transferrin receptor was also found to be overexpressed in the superinvasive cell line. Conclusion: Cleavage of serum proteins such as transferrin could provide a valuable source of markers for malignant tumours and could also play a role in aspects of cancer pathogenesis, such as tumour cachexia

Identification of pancreatic cancer invasion-related proteins by proteomic analysis

Background – Markers of pancreatic cancer invasion were investigated in two clonal populations of the cell line, MiaPaCa-2, Clone #3 (high invasion) and Clone #8 (low invasion) using proteomic profiling of an in vitro model of pancreatic cancer. Materials and methods – Using 2D-DIGE followed by MALDI-TOF MS, two clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with high and low invasive capacities were incubated on matrigel 24 hours prior to analysis to stimulate cell-ECM contact and mimic in vivo interaction with the basement membrane. Results - Sixty proteins were identified as being differentially expressed (>1.2 fold change and p ≤ 0.05) between Clone #3 and Clone #8. Proteins found to have higher abundance levels in the highly invasive Clone #3 compared to the low invasive Clone #8 include members of the chaperone activity proteins and cytoskeleton constituents whereas metabolism-associated and catalytic proteins had lower abundance levels. Differential protein expression levels of ALDH1A1, VIM, STIP1 and KRT18 and GAPDH were confirmed by immunoblot. Using RNAi technology, STIP1 knockdown significantly reduced invasion and proliferation of the highly invasive Clone #3. Knockdown of another target, VIM by siRNA in Clone #3 cells also resulted in decreased invasion abilities of Clone #3. Elevated expression of STIP1 was observed in pancreatic tumour tissue compared to normal pancreas, whereas ALDH1A1 stained at lower levels in pancreatic tumours, as detected by immunohistochemistry. Conclusion - Identification of targets which play a role in the highly invasive phenotype of pancreatic cancer may help to understand the biological behaviour, the rapid progression of this cancer and may be of importance in the development of new therapeutic strategies for pancreatic cancer

Induction of apoptosis in yeast and mammalian cells by exposure to 1,10-phenanthroline metal complexes

1,10-Phenanthroline (phen) and metal–phen complexes display fungicidal and fungiststic activity, disrupt mitochondrial function and induce oxidative stress. We have examined the effect of these drugs on the structure of yeast and mammalian cell organelles and the integrity of cellular DNA. Exposure of Candida albicans to [Mn(phen)2(mal)].2H2O or [Ag2(phen)3(mal)].2H2O (mal H2=malonic acid) resulted in DNA degradation whereas exposure to phen or [Cu(phen)2(mal)].2H2O did not. All drugs induced extensive changes to the internal structure of yeast cells including retraction of the cytoplasm, nuclear fragmentation and disruption of the mitochondrion. In the case of cultured mammalian cells [Cu(phen)2(mal)].2H2O induced apoptosis as evidenced by the ladder pattern of DNA fragments following gel electrophoresis and also the blebbing of the cell membrane. The other drugs produced non-specific DNA degradation in mammalian cells. In conclusion, phen and metal–phen complexes have the potential to induce apoptosis in fungal and mammalian cells. Given their distinct mode of action compared to conventional anti-fungal drugs, phen and metal–phen complexes may represent a novel group of anti-fungal agents for use either in combination with existing drugs or in cases where resistance to conventional drugs has emerged

Induction of apoptosis in yeast and mammalian cells by exposure to 1,10-phenanthroline metal complexes

1,10-Phenanthroline (phen) and metal–phen complexes display fungicidal and fungiststic activity, disrupt mitochondrial function and induce oxidative stress. We have examined the effect of these drugs on the structure of yeast and mammalian cell organelles and the integrity of cellular DNA. Exposure of Candida albicans to [Mn(phen)2(mal)].2H2O or [Ag2(phen)3(mal)].2H2O (mal H2=malonic acid) resulted in DNA degradation whereas exposure to phen or [Cu(phen)2(mal)].2H2O did not. All drugs induced extensive changes to the internal structure of yeast cells including retraction of the cytoplasm, nuclear fragmentation and disruption of the mitochondrion. In the case of cultured mammalian cells [Cu(phen)2(mal)].2H2O induced apoptosis as evidenced by the ladder pattern of DNA fragments following gel electrophoresis and also the blebbing of the cell membrane. The other drugs produced non-specific DNA degradation in mammalian cells. In conclusion, phen and metal–phen complexes have the potential to induce apoptosis in fungal and mammalian cells. Given their distinct mode of action compared to conventional anti-fungal drugs, phen and metal–phen complexes may represent a novel group of anti-fungal agents for use either in combination with existing drugs or in cases where resistance to conventional drugs has emerged

Proteomic screening of glucose-responsive and glucose non-responsive MIN-6 beta cells reveals differential expression of proteins involved in protein folding, secretion and oxidative stress

The glucose-sensitive insulin-secretion (GSIS) phenotype is relatively unstable in long-term culture of beta cells. The purpose of this study was to investigate relative changes in the proteome between glucose-responsive (low passage) and glucose non-responsive (high passage) murine MIN-6 pancreatic beta cells. The 2D-DIGE and subsequent DeCyder analysis detected 3351 protein spots in the pH range of 4–7. Comparing MIN-6(H) to MIN-6(L) and using a threshold of 1.2-fold, the number of proteins with a decrease in expression level was 152 (4.5%), similar was 3140 (93.7%) and increased 59 (1.8%). From the differentially expressed proteins identified in this study, groups of proteins associated with the endoplasmic reticulum (ER) and proteins involved in oxidative stress were found to be significantly decreased in the high-passage (H passage) cells. These proteins included endoplasmic reticulum protein 29 (ERp29); 78-kDa glucoserelated protein, (GRP78); 94-kDa glucose-related protein (GRP94); protein disulphide isomerase; carbonyl reductase 3; peroxidoxin 4 and superoxide dismutase 1. These results suggest that non- GSIS MIN-6 cells do not have the same ability/capacity of glucose-responsive MIN-6 cells to successfully fold, modify or secrete proteins and counteract the problems associated with oxidative stress

Yeast mannan-rich fraction modulates endogenous reactive oxygen species generation and antibiotic sensitivity in resistant E. coli

Mannan-rich fraction (MRF) isolated from Saccharomyces cerevisiae has been studied for its beneficial impact on animal intestinal health. Herein, we examined how MRF affected the formation of reactive oxygen species (ROS), impacting antibiotic susceptibility in resistant Escherichia coli through the modulation of bacterial metabolism. The role of MRF in effecting proteomic change was examined using a proteomics-based approach. The results showed that MRF, when combined with bactericidal antibiotic treatment, increased ROS production in resistant E. coli by 59.29 ± 4.03% compared to the control (p ≤ 0.05). We further examined the effect of MRF alone and in combination with antibiotic treatment on E. coli growth and explored how MRF potentiates bacterial susceptibility to antibiotics via proteomic changes in key metabolic pathways. Herein we demonstrated that MRF supplementation in the growth media of ampicillin-resistant E. coli had a significant impact on the normal translational control of the central metabolic pathways, including those involved in the glycolysis–TCA cycle (p ≤ 0.05