Jill A. Opsahl

Iodine concentration in Norwegian milk and dairy products

The present study was conducted to determine the iodine concentration in Norwegian-produced milk and a selection of dairy products. The iodine concentration of eighty-five samples of milk and dairy products was analysed by inductively coupled plasma-MS. Low-fat milk and organic milk were sampled from nineteen and seven different locations in Norway, respectively, during the summer and winter season of 2000. Other milk and dairy products were chiefly collected during the summer season. Low-fat milk from the summer season had significantly lower median iodine concentration (88 microg/l, range 63-122 microg/l) compared with low-fat milk from the winter season (232 microg/l, range 103-272 microg/l). The median iodine concentration of organic summer milk (60 microg/l) was significantly lower than the iodine concentration of organic winter milk (127 microg/l). There were no significant differences in the low-fat-milk samples with regard to geographical sampling location. Whey cheese (Tine Gudbrandsdalsost) iodine concentration was significantly higher (803 microg/kg) than the median iodine concentration in casein cheeses such as Jarlsberg and Norvegia of 201 and 414 microg/kg, respectively. With a recommended iodine intake of 150 microg/d for adults, a daily intake of 0.4 litres milk meets the requirement with 25 % during the summer and more than 60 % during the winter season. Thus, milk and dairy products are important determinants of iodine intake in Norway.

Proteomics of human cerebrospinal fluid: Discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics

There is an urgent need for novel biomarkers that can be used to improve the diagnosis, predict the disease progression, improve our understanding of the pathology or serve as therapeutic targets for neurodegenerative diseases. Cerebrospinal fluid (CSF) is in direct contact with the CNS and reflects the biochemical state of the CNS under different physiological and pathological settings. Because of this, CSF is regarded as an excellent source for identifying biomarkers for neurological diseases and other diseases affecting the CNS. Quantitative proteomics and sophisticated computational software applied to analyze the protein content of CSF has been fronted as an attractive approach to find novel biomarkers for neurological diseases. This review will focus on some of the potential pitfalls in biomarker studies using CSF, summarize the status of the field of CSF proteomics in general, and discuss some of the most promising proteomics biomarker study approaches. A brief status of the biomarker discovery efforts in multiple sclerosis, Alzheimers disease, and Parkinsons disease is also given.

In-depth Characterization of the Cerebrospinal Fluid (CSF) Proteome Displayed Through the CSF Proteome Resource (CSF-PR)

In this study, the human cerebrospinal fluid (CSF) proteome was mapped using three different strategies prior to Orbitrap LC-MS/MS analysis: SDS-PAGE and mixed mode reversed phase-anion exchange for mapping the global CSF proteome, and hydrazide-based glycopeptide capture for mapping glycopeptides. A maximal protein set of 3081 proteins (28,811 peptide sequences) was identified, of which 520 were identified as glycoproteins from the glycopeptide enrichment strategy, including 1121 glycopeptides and their glycosylation sites. To our knowledge, this is the largest number of identified proteins and glycopeptides reported for CSF, including 417 glycosylation sites not previously reported. From parallel plasma samples, we identified 1050 proteins (9739 peptide sequences). An overlap of 877 proteins was found between the two body fluids, whereas 2204 proteins were identified only in CSF and 173 only in plasma. All mapping results are freely available via the new CSF Proteome Resource (http://probe.uib.no/csf-pr), which can be used to navigate the CSF proteome and help guide the selection of signature peptides in targeted quantitative proteomics.

Cerebrospinal fluid proteome comparison between multiple sclerosis patients and controls.

The aim of the present study was to identify proteins in cerebrospinal fluid (CSF) with different abundance between patients with relapsing‐remitting multiple sclerosis (RRMS) and controls. Such proteins may be diagnostic biomarkers and contribute with novel information about the disease pathogenesis.

Effects of blood contamination and the rostro-caudal gradient on the human cerebrospinal fluid proteome.

Over the last years there has been an increased focus on the importance of knowing the effect of pre-analytical influence on the proteomes under study, particularly in the field of biomarker discovery. We present three proteomics studies examining the effect of blood contamination and the rostro-caudal gradient (RCG) on the cerebrospinal fluid (CSF) proteome, in addition to plasma/CSF protein ratios. The studies showed that the central nervous system (CNS) derived proteins appeared to be unaffected by the RCG, while the plasma-derived proteins showed an increase in concentration towards the lumbar area. This implies that the concentration of the plasma-derived proteins in CSF will vary depending on the volume of CSF that is collected. In the CSF samples spiked with blood, 262 of 814 quantified proteins showed an abundance increase of more than 1.5 fold, while 403 proteins had a fold change of less than 1.2 and appeared to be unaffected by blood contamination. Proteins with a high plasma/CSF ratio appeared to give the largest effect on the CSF proteome upon blood contamination. The results give important background information on how factors like blood contamination, RCG and blood-CNS-barrier influences the CSF proteome. This information is particularly important in the field of biomarker discovery, but also for routine clinical measurements. The data from the blood contamination and RCG discovery studies have been deposited to the ProteomeXchange with identifier PXD000401.

Iodine intake and status in two groups of Norwegians

Objective: To evaluate the ranges of iodine intake and iodine status in two subgroups of the Norwegian population. Design: The participants in the Tromso group (n=63) had a normal diet. The participants in the Bergen group (n=44) comprised people with a variable intake of fish and dairy products. Results: The iodine intake varied from 56 to 318 mg day-1 in the Tromso group and from 30 to 427 mg day–1 in the Bergen group. Median intake of iodine (162 µg day-1) in the Tromso group was significantly higher than in the Bergen group (89 µg day-1) (<p/0.001). Urinary iodine concentration varied from 38 to 572 µg l-1 with a median value of 117 µg l-1 in the Tromso group. Median iodine excretion in the Bergen group was 96 µg 24 h-1 (85 µg l-1) and varied from 16 to 316 µg 24 h-1. Median serum thyroid-stimulating hormone was 1.6 mIU l-1 in the Tromso group and 1.4 mIU l-1 in the Bergen group. Median free thyroxine in serum was significantly higher in the p articipants from Tromso (15 pmol l-1) than in those from Bergen (12 pmol l-1). Conclusions: Regular intake of dairy products and/or fish products is important to meet the iodine requirements in the Norwegian diet. Although the iodine intake was found to be in the intake range of sufficient iodine intake of most of the participants in the two groups, there may exist population groups with a very low average intake of iodine. Keywords: Diet; iodine; iodine excretion; Norwegians; thyroid hormones

Cerebrospinal fluid proteomics in multiple sclerosis.

Multiple sclerosis (MS) is an immune mediated chronic inflammatory disease of the central nervous system usually initiated during young adulthood, affecting approximately 2.5 million people worldwide. There is currently no cure for MS, but disease modifying treatment has become increasingly more effective, especially when started in the first phase of the disease. The disease course and prognosis are often unpredictable and it can be challenging to determine an early diagnosis. The detection of novel biomarkers to understand more of the disease mechanism, facilitate early diagnosis, predict disease progression, and find treatment targets would be very attractive. Over the last decade there has been an increasing effort toward finding such biomarker candidates. One promising strategy has been to use state-of-the-art quantitative proteomics approaches to compare the cerebrospinal fluid (CSF) proteome between MS and control patients or between different subgroups of MS. In this review we summarize and discuss the status of CSF proteomics in MS, including the latest findings with a focus on the last five years. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia

BackgroundThe small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy.ResultsNutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells.ConclusionsAltogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML.

Identification of Dynamic Changes in Proteins Associated with the Cellular Cytoskeleton after Exposure to Okadaic Acid

Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100–500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment.

Increased interaction between DJ-1 and the Mi-2/ nucleosome remodelling and deacetylase complex during cellular stress

DJ‐1 was originally identified to be an oncogenic product, but has later been shown to be highly multifunctional. DJ‐1 plays a role in oxidative stress response and transcriptional regulation, and loss of its function leads to an early onset of Parkinsonism. To further understand the mechanisms behind DJ‐1s role in cell survival and death, we investigated alternations in endogenous DJ‐1 protein–protein interaction in apoptotic cells exposed to the phosphatase inhibitor okadaic acid. By combining cellular stable isotopic labelling of amino acids in cell culture, sub‐cellular fractionation, co‐immunoprecipitation, and MS, we identified a novel group of DJ‐1 interaction partners that increased their association to DJ‐1 in okadaic acid‐exposed cells. These proteins were integral components of the Mi‐2/nucleosome remodelling and deacetylase (NuRD) complex. Knockdown of DJ‐1 and MTA2, a core component of the NuRD complex, had a similar and pro‐apoptotic effect on the transcriptional‐ and p53‐dependent cell death induced by daunorubicin. On the other hand, MTA2 knockdown had no significant effect on the progression of p53‐independent okadaic acid‐induced apoptosis. Our data suggest that the increased DJ‐1/NuRD interaction is a general anti‐stress response regulated by okadaic acid‐induced modifications of DJ‐1. The observed interaction between DJ‐1 and the NuRD complex may give new clues to how DJ‐1 can protect cells from p53‐dependent cell death.