RNA modifications by oxidation: A novel disease mechanism?

The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas other RNA molecules show virtually no oxidation. The iron-storage disease hemochromatosis exhibits the most prominent general increase in RNA oxidation ever observed. Oxidation of RNA primarily leads to strand breaks and to oxidative base modifications. Oxidized mRNA is recognized by the ribosomes, but the oxidation results in ribosomal stalling and dysfunction, followed by decreased levels of functional protein as well as the production of truncated proteins that do not undergo proper folding and may result in protein aggregation within the cell. Ribosomal dysfunction may also signal apoptosis by p53-independent pathways. There are very few reports on interventions that reduce RNA oxidation, one interesting observation being a reduction in RNA oxidation by ingestion of raw olive oil. High urinary excretion of 8-oxo-guanosine, a biomarker for RNA oxidation, is highly predictive of death in newly diagnosed type 2 diabetics; this demonstrates the clinical relevance of RNA oxidation. Taken collectively the available data suggest that RNA oxidation is a contributing factor in several diseases such as diabetes, hemochromatosis, heart failure, and β-cell destruction. The mechanism involves free iron and hydrogen peroxide from mitochondrial dysfunction that together lead to RNA oxidation that in turn gives rise to truncated proteins that may cause aggregation. Thus RNA oxidation may well be an important novel contributing mechanism for several diseases.     

Cholangiocytes in the pathogenesis of primary sclerosing cholangitis and development of cholangiocarcinoma

Primary sclerosing cholangitis (PSC) is an idiopathic cholangiopathy strongly associated with inflammatory bowel disease (IBD) and characterized by cholestasis, chronic immune infiltration and progressive fibrosis of the intrahepatic and extrahepatic bile ducts. PSC confers a high risk of cholangiocarcinoma (CCA) with PSC-CCA representing the leading cause of PSC-associated mortality. PSC-CCA is derived from cholangiocytes and associated progenitor cells – a heterogeneous group of dynamic epithelial cells lining the biliary tree that modulate the composition and volume of bile production by the liver. Infection, inflammation and cholestasis can trigger cholangiocyte activation leading to an increased expression of adhesion and antigen-presenting molecules as well as the release of various inflammatory and fibrogenic mediators. As a result, activated cholangiocytes engage in a myriad of cellular processes, including hepatocellular proliferation, apoptosis, angiogenesis and fibrosis. Cholangiocytes can also regulate the recruitment of immune cells, mesenchymal cells, and endothelial cells that participate in tissue repair and destruction in settings of persistent inflammation. In PSC, the role of cholangiocytes and the mechanisms governing their transformation to PSC-CCA are unclear however localization of disease suggests that cholangiocytes are a key target and potential regulator of hepatobiliary immunity, fibrogenesis and tumorigenesis. Herein, we summarize mechanisms of cholangiocyte activation in PSC and highlight new insights into disease pathways that may contribute to the development of PSC-CCA. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Bi-modal vertical distribution of the carnivorous copepod Paraeuchaeta norvegica

Bi-modal vertical distribution of Paraeuchaeta norvegica duringlate autumn was observed in a Norwegian fjord in three differentyears, and hence seems to be a recurring phenomenon. An uppermode performed diel vertical migration, foraging in shallowwater at night. A deep mode did not migrate vertically, butsome feeding took place at depth.     

Prevalence of skull pathologies in European harbor seals (<Emphasis Type="Italic">Phoca vitulina</Emphasis>) during 1981–2014

Harbor seals (Phoca vitulina) inhabit the seas surrounding Denmark and are an important top predator in the marine food chain. This trophic position exposes them to environmental contaminants with disease epidemics and hunting being additional threats to this population. It is therefore important to study how environmental pollution at the current order of magnitude affects the health of the population. Earlier studies have shown that occurrence of periodontitis could be linked to the amount of pollution the seals were subjected to. In order to investigate this further, 380 skulls and 141 mandibles of harbor seals (Phoca vitulina) from the Wadden Sea, the Limfjord, and Kattegat collected during the period 1970–2014 were examined. The skulls were examined for pathological lesions. The Hounsfield Units (HU) which are correlated to the bone mineral density (BMD) were measured in a subsample (n = 34) using CT scans. The macroscopic examination revealed (with the exception of the Swedish part of Kattegat) a significant increase of pathological lesions over the study period of 1981–2014. The examination of HU showed that median HU measured at multiple sites was highest in the healthy skulls compared to the skulls with one or more of the lesions. A discriminant analysis allowed high discriminatory capacity to separate healthy skulls from the skulls with pathologies, simply by the utilization of the HU data. Former studies of BMD in marine mammals have shown that exposure to environmental chemicals alter BMD and cause periodontitis. The present study, based on temporal and spatial trends in BMD, confirms the results of previous studies.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.     

Bim and Bcl-2 mutually affect the expression of the other in T cells

The life and death of T cells is controlled to a large extent by the relative amounts of Bcl-2-related proteins they contain. The antiapoptotic protein Bcl-2 and the proapoptotic protein Bim are particularly important in this process with the amount of Bcl-2 per cell dropping by about one-half when T cells prepare to die. In this study we show that Bcl-2 and Bim each control the expression of the other. Absence of Bim leads to a drop in the amount of intracellular Bcl-2 protein, while having no effect on the amounts of mRNA for Bcl-2. Conversely, high amounts of Bcl-2 per cell allow high amounts of Bim, although in this case the effect involves increases in Bim mRNA. These mutual effects occur even if Bcl-2 is induced acutely. Thus these two proteins control the expression of the other, at either the protein or mRNA level.     

Age- and Sex-Associated Effects on Acute-Phase Proteins in G?ttingen Minipigs

Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP in Göttingen minipigs and to investigate the effects of age, sex, and various stimuli on these ranges. Serum concentrations of C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, pig major acute-phase protein (PMAP), albumin, and porcine α-1 acid glycoprotein (PAGP) were evaluated in 4 age groups (6, 16, 24 and 40–48 wk) of male and female Göttingen minipigs. In addition, minipigs were tested under 2 housing conditions, after acute LPS challenge, and after diet-induced obesity with and without mild diabetes. Changing the pigs to a new environment induced significant increases in CRP, PMAP, haptoglobin and PAGP and a decrease in albumin. An acute LPS stimulus increased CRP, PMAP, haptoglobin, and SAA; PAGP was unchanged and albumin decreased. Obese pigs with and without diabetes showed increases in CRP and PAGP, albumin decreased, and haptoglobin and SAA were unchanged. PMAP was increased only in obese pigs without diabetes. In conclusion, reference values for CRP, PMAP, haptoglobin, SAA, PAGP and albumin were established for male and female Göttingen minipigs of different ages. These APP were influenced by age and sex, underlining the importance of considering these factors when designing and interpreting studies including aspects of inflammation. In addition, an APP response was verified after both acute and chronic stimuli. Abbreviations: APP, acute-phase proteins; APR, acute-phase response; CRP, C-reactive protein; HFD, high-fat diet; HFD+D, high fat diet + diabetes; PAGP, porcine α1 acid glycoprotein; PMAP, pig major acute-phase protein; SAA, serum amyloid AInflammation is involved in a number of important and increasingly widespread human diseases, including inflammatory bowel diseases, cancers, infections, metabolic diseases like obesity and diabetes, and cardiovascular diseases like atherosclerosis.^{1,5,7,11,20,41} The systemic response to inflammation is the acute-phase response (APR) which, together with innate immune responses, prevents infection, clears pathogens, and contributes to inflammation resolution and the healing process. The APR has been extensively described in humans^10,22 and other mammals,^8,14,29,31 and in all cases, it is regulated by cytokines including IL6 and TNFα.^21,30 The APR is activated by many different stimuli, including trauma, infection, stress, neoplasia, and inflammatory stimuli, resulting in significant changes in the circulating concentrations of the so-called acute-phase proteins (APP). The APP are synthetized primarily by the liver and can be divided into positive and negative APP depending on whether their concentration in plasma increases (positive) or decreases (negative) in response to a stimulus.¹⁰ In addition, they can be divided into major and minor APP, depending on the magnitude of their concentration change after a given stimulus.²² Because the concentrations of the APP change in response to a given stimulus, their serum or plasma levels can be used diagnostically as biomarkers of disease severity and progression or to evaluate the effect of various interventions.^8,14,31 The APP show different kinetics after a stimulus, with C-reactive protein (CRP) and serum amyloid A (SAA) displaying rapid increases and normalization after the stimulus has been removed, whereas haptoglobin shows a later and more prolonged response.^10,31 The APR may be transient and revert to normal with recovery, or it can persist, as during chronic conditions.²¹ Importantly, APP and their kinetics differ somewhat between species.³¹To further elucidate the involvement of inflammation in human diseases, accurate animal models of inflammation, including species validated biomarkers of inflammation, are needed. Mouse models are commonly used in many research areas, but their response to several different inflammatory conditions is not comparable to that of humans, and therefore the predictive validity of these models may be limited.³⁹ Pigs are highly comparable to humans with respect to anatomy and physiology,⁴⁴ and their APR to various stimuli has been described.^14,23,26 In general, the APR and the resulting changes in APP seem to be very similar in pigs compared with humans, with CRP, haptoglobin, and SAA being major positive APP and albumin being a negative APP.¹⁴ In humans, α1-acid glycoprotein (AGP) is a positive APP but has been reported to either increase,¹⁷ remain unchanged^23,45 or to decrease¹² in pigs, depending on the stimulus investigated. The concentrations of some of the major APP characterized in domestic pigs show significant effects of age and sex.^32,34 In addition to age and sex effects, significant differences in APP between herds have been observed, most likely reflecting different pathogenic pressures in the different herds.³² Furthermore, some indications exist for possible interbreed differences in APP concentrations, although this possibility has not been investigated in detail.¹²Minipigs are especially relevant in biomedical research, given their smaller size and well-defined microbiology and genetics.⁴ Göttingen minipigs are a useful model for several conditions involving inflammation and the APR, including infection,² obesity,¹⁹ diabetes²⁴ and atherosclerosis,¹⁸ and different APP have already been used as biomarkers in some of these models.² Therefore, existing data suggest that APP commonly applied in human medicine could be relevant in Göttingen minipigs as well. However, the APR and reference values of APP, including the potential influence of age and sex indicated in other studies, have not been investigated systematically in this breed.^12,32,34The objective of the current study was to establish reference values of selected APP in normal Göttingen minipigs, including evaluation of the possible effects of age and sex. In addition, the effects of housing condition and acute and chronic inflammatory stimuli were assessed.     

A magnetic adsorbent-based process for semi-continuous PEGylation of proteins

A semi-continuous magnetic particle-based process for the controlled attachment of PEG (PEGylation) to proteins is described for the first time. Trypsin and 2 kDa mono-activated PEG were used to systematically develop the steps in the process. Proof of concept was shown in a microfluidics system to minimize reagent consumption. Two streams containing (i) 1.2 g/L trypsin and (ii) 4 g/L magnetic adsorbents derivatized with the reversible affinity ligand benzamidine were pumped into a pipe reactor. At the exit, a third solution of activated PEG (0-40 g/L) was introduced and the solutions immediately fed into a second reactor. Upon exiting, the mixture was combined in a third reactor with a fourth stream of free amine groups to stop the reaction (50 mM lysine). The mixture continued into a high-gradient magnetic separator where magnetic supports, with PEGylated trypsin still attached, were captured and washing and elution steps were subsequently carried out. Analysis of the conjugates (with SDS-PAGE & LC-MS) showed that the extent of PEGylation could be controlled by varying the reaction time or PEG concentration. Furthermore, the PEG-conjugates had higher enzyme activity compared to PEGylation of non-immobilized trypsin.