Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

Background

The photorespiratory nitrogen cycle in C₃ plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO₂. Because of the loss of CO₂ and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C₃ plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 4.1.1.47) and hydroxypyruvate isomerase (EC 5.3.1.22) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle.

Results

When grown in ambient air, but not in elevated CO₂, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation.

Conclusions

Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a deleterious short-circuit of the photorespiratory nitrogen cycle. This diversion in metabolism gave rise to increased concentrations of amino acids, in particular glutamine and asparagine in the leaves and a decrease of soluble sugars.     

A GWAS assessment of the contribution of genomic imprinting to the variation of body mass index in mice

Background

Genomic imprinting is an epigenetic mechanism that can lead to differential gene expression depending on the parent-of-origin of a received allele. While most studies on imprinting address its underlying molecular mechanisms or attempt at discovering genomic regions that might be subject to imprinting, few have focused on the amount of phenotypic variation contributed by such epigenetic process. In this report, we give a brief review of a one-locus imprinting model in a quantitative genetics framework, and provide a decomposition of the genetic variance according to this model. Analytical deductions from the proposed imprinting model indicated a non-negligible contribution of imprinting to genetic variation of complex traits. Also, we performed a whole-genome scan analysis on mouse body mass index (BMI) aiming at revealing potential consequences when existing imprinting effects are ignored in genetic analysis.

Results

10,021 SNP markers were used to perform a whole-genome single marker regression on mouse BMI using an additive and an imprinting model. Markers significant for imprinting indicated that BMI is subject to imprinting. Marked variance changed from 1.218 ×10⁻⁴ to 1.842 ×10⁻⁴ when imprinting was considered in the analysis, implying that one third of marked variance would be lost if existing imprinting effects were not accounted for. When both marker and pedigree information were used, estimated heritability increased from 0.176 to 0.195 when imprinting was considered.

Conclusions

When a complex trait is subject to imprinting, using an additive model that ignores this phenomenon may result in an underestimate of additive variability, potentially leading to wrong inferences about the underlying genetic architecture of that trait. This could be a possible factor explaining part of the missing heritability commonly observed in genome-wide association studies (GWAS).     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

An improved formulation of the zirconyl hematoxylin stain for acidic mucins

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.     

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

Background  

Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4⁺ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD).     

Methods for peptide identification by spectral comparison

Background  

Tandem mass spectrometry followed by database search is currently the predominant technology for peptide sequencing in shotgun proteomics experiments. Most methods compare experimentally observed spectra to the theoretical spectra predicted from the sequences in protein databases. There is a growing interest, however, in comparing unknown experimental spectra to a library of previously identified spectra. This approach has the advantage of taking into account instrument-dependent factors and peptide-specific differences in fragmentation probabilities. It is also computationally more efficient for high-throughput proteomics studies.     

Coping styles and behavioural flexibility: towards underlying mechanisms

A coping style (also termed behavioural syndrome or personality) is defined as a correlated set of individual behavioural and physiological characteristics that is consistent over time and across situations. This relatively stable trait is a fundamental and adaptively significant phenomenon in the biology of a broad range of species, i.e. it confers differential fitness consequences under divergent environmental conditions. Behavioural flexibility appears to be an important underlying attribute or feature of the coping style that might explain consistency across situations. Proactive coping is characterized by low flexibility expressed as rather rigid, routine-like behavioural tendencies and reduced impulse control (behavioural inhibition) in operant conditioning paradigms. This article summarizes some of the evidence that individual differentiation in behavioural flexibility emerges as a function of underlying variability in the activation of a brain circuitry that includes the prefrontal cortex and its key neurochemical signalling pathways (e.g. dopaminergic and serotonergic input). We argue that the multidimensional nature of animal personality and the terminology used for the various dimensions should reflect the differential pattern of activation of the underlying neuronal network and the behavioural control function of its components. Accordingly, unravelling the molecular mechanisms that give rise to individual differences in the coping style will be an important topic in biobehavioural neurosciences, ecology and evolutionary biology.