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Additional file 3: Fig. E3. SDS-PAGE and immunoblot is shown for both bovine collagen after duodenal digestion preceded by gastric digestion at pH 1.2/PPR 0.1. Under these conditions, bovine collagen is fully resistant to duodenal digestion.

Additional file 1: Fig. E1. Selected SDS-PAGE and immunoblot samples are shown for both albumins, Ara h 2 and pea albumin PA2. Panel a illustrates that pea albumin PA2 is truncated at low PPR but truncated molecules are still detected on immunoblot. Panel b shows gastric digestion of Ara h 2 at low pH and high PPR. Under these conditions the upper band is quite stable but the lower band is truncated. Truncated molecules are not recognized on immunoblot. Panel c and d illustrates that Ara h 2 (panel c) and pea albumin PA2 (panel d) behave differently when gastric digestion at pH 1.2 and pH 2.5 are compared. Ara h 2 is quite stable under both conditions, but if anything slightly more susceptible to digestion at pH 2.5. For pea albumin PA2 this is the other way around. It is clearly more resistant to digestion at pH 2.5 than pH 1.2.

Discussion of organelle inheritance

Discussion of establishment of sex in multicellular organisms

Discussion of ROS producing processes during photosynthesis

Vanishing white matter (VWM) is a leukodystrophy with predominantly early-childhood onset. Affected children display various neurological signs, including ataxia and spasticity, and die early. VWM patients have bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (eIF2B). eIF2B regulates protein synthesis rates under basal and cellular stress conditions. The underlying molecular mechanism of how mutations in eIF2B result in VWM is unknown. Previous studies suggest that brain white matter astrocytes are primarily affected in VWM. We hypothesized that the translation rate of certain astrocytic mRNAs is affected by the mutations, resulting in astrocytic dysfunction. Here we subjected primary astrocyte cultures of wild type (wt) and VWM (2b5ho) mice to pulsed labeling proteomics based on stable isotope labeling with amino acids in cell culture (SILAC) with an L-azidohomoalanine (AHA) pulse to select newly synthesized proteins. AHA was incorporated into newly synthesized proteins in wt and 2b5ho astrocytes with similar efficiency, without affecting cell viability. We quantified proteins synthesized in astrocytes of wt and 2b5ho mice. This proteomic profiling identified a total of 80 proteins that were regulated by the eIF2B mutation.We confirmed increased expression of PROS1 in 2b5ho astrocytes and brain. A DAVID enrichment analysis showed that approximately 50% of the eIF2B-regulated proteins used the secretory pathway. A small-scale metabolic screen further highlighted a significant change in the metabolite 6-phospho-gluconate, indicative of an altered flux through the pentose phosphate pathway (PPP). Some of the proteins migrating through the secretory pathway undergo oxidative folding reactions in the endoplasmic reticulum (ER), which produces reactive oxygen species (ROS). The PPP produces NADPH to remove ROS. The proteomic and metabolomics data together suggest a deregulation of ER function in 2b5ho mouse astrocytes.

Organotypic cultures from normal neocortical tissue obtained at epilepsy surgery show a severe injury response. This response involves both neuronal degeneration and the proliferation of reactive cells. A salient feature of the reactive cells is the co-expression of microglial and astrocytic markers. Surprisingly, the reactive cells also began to express neuronal markers Tubulin βIII and MAP2 adding to the confusion about their origin. Concomitant with their appearance in reactive cells MAP2 and Tubulin βIII expression disappeared from neurons. While NeuN expression decreased significantly, it did not entirely disappear from many neurons. Moreover, it was not observed in reactive cells, showing that NeuN is a reliable marker of neurons.

Brain injury affects a significant number of people each year. Organotypic cultures from resected normal neocortical tissue provide unique opportunities to study the cellular and neuropathological consequences of severe injury of adult human brain tissue in vitro. The in vitro injuries caused by resection (interruption of the circulation) and aggravated by the preparation of slices (severed neuronal and glial processes and blood vessels) reflect the reaction of human brain tissue to severe injury. We investigated this process using immunocytochemical markers, reverse transcriptase quantitative polymerase chain reaction and Western blot analysis. Essential features were rapid shrinkage of neurons, loss of neuronal marker expression and proliferation of reactive cells that expressed Nestin and Vimentin. Also, microglia generally responded strongly, whereas the response of glial fibrillary acidic protein-positive astrocytes appeared to be more variable. Importantly, some reactive cells also expressed both microglia and astrocytic markers, thus confounding their origin. Comparison with post-mortem human brain tissue obtained at rapid autopsies suggested that the reactive process is not a consequence of epilepsy.

The “RNA world” hypothesis is seen as one of the main contenders for a viable theory on the origin of life. Relatively small RNAs have catalytic power, RNA is everywhere in present-day life, the ribosome is seen as a ribozyme, and rRNA and tRNA are crucial for modern protein synthesis. However, this view is incomplete at best. The modern protein-RNA ribosome most probably is not a distorted form of a “pure RNA ribosome” evolution started out with. Though the oldest center of the ribosome seems “RNA only”, we cannot conclude from this that it ever functioned in an environment without amino acids and/or peptides. Very small RNAs (versatile and stable due to basepairing) and amino acids, as well as dipeptides, coevolved. Remember, it is the amino group of aminoacylated tRNA that attacks peptidyl-tRNA, destroying the bond between peptide and tRNA. This activity of the amino acid part of aminoacyl-tRNA illustrates the centrality of amino acids in life. With the rise of the “RNA world” view of early life, the pendulum seems to have swung too much towards the ribozymatic part of early biochemistry. The necessary presence and activity of amino acids and peptides is in need of highlighting. In this article, we try to bring the role of the peptide component of early life back into focus. We argue that an RNA world completely independent of amino acids never existed.

The gene prmC, encoding the putative S-adenosyl-L-methionine (AdoMet) -dependent methyltransferase (MTase) of release factors (RFs) of the obligate intracellular pathogen Chlamydia trachomatis, was functionally analyzed. Chlamydial PrmC expression suppresses the growth defect of a prmC knockout strain of Escherichia coli K-12, suggesting an interaction of chlamydial PrmC with E. coli RFs in vivo. In vivo methylation assays carried out with recombinant PrmC and RFs of chlamydial origin demonstrated that PrmC methylates RFs within the tryptic fragment containing the universally conserved sequence motif Gly-Gly-Gln. This is consistent with the enzymatic properties of PrmC of E. coli origin. We conclude that C. trachomatis PrmC functions as an N-5-glutamine AdoMet-dependent MTase, involved in methylation of RFs

In this work, computational tools are used to investigate three issues in evolutionary biochemistry. After an introductory chapter, chapter 2 searches for short, ancient motives in contemporary proteins by focusing on the metal-binding character of aspartic acid; three such motives were found. The next issue is the structure of the genetic code. Refinements to the existing knowledge are contributed in chapter 3. The code belonging to the global optimum for error minimization in a widely-used code space (a long-standing question in the field) is determined by an exact method. Larger code spaces are formulated mathematically and investigated with an improved error function. This raises the debate over which code space should be used in this kind of studies. Most importantly, published possible scenarios concerning the evolution of the genetic code are shown to be all (including Crick’s Frozen Accident Theory) consistent with error-robust structure of the code. In chapter 4, the focus is on similar code triplets encoding the same amino acid. A hitherto unknown regularity in the standard genetic code is presented: No canonical amino acid is encoded by one single A-starting codon only, and this regularity, in combination with the wobble behavior of tRNAs with unmodified G-starting and C-starting codons, has implications for likely primordial tRNA sets. Chapter 5 introduces values to study the genetic code regularities with respect to molecular structure similarity of amino acids. In the final chapter, pan-editing in trypanosomes is investigated using computational tools.