Biological Evaluations, NMR Analyses, Molecular Modeling Studies, and Overview of the Synthesis of the Marine Natural Product (-)-Mucosin.

Natural products obtained from marine organisms continue to be a rich source of novel structural architecture and of importance in drug discovery, medicine, and health. However, the success of such endeavors depends on the exact structural elucidation and access to sufficient material, often by stereoselective total synthesis, of the isolated natural product of interest. (-)-Mucosin (1), a fatty acid derivative, previously presumed to contain a rare cis-bicyclo[4.3.0]non-3-ene moiety, has since been shown to be the trans-congener. Analytically, the fused bicyclic ring system in (-)-1 constitutes a particular challenge in order to establish its relative and absolute stereochemistry. Herein, data from biological evaluations, NMR and molecular modeling studies of (-)-1 are presented. An overview of the synthetic strategies enabling the exact structural elucidation of (-)-mucosin (1) is also presented.

Glycated Proteins, Glycine, Acetate, and Monounsaturated Fatty Acids May Act as New Biomarkers to Predict the Progression of Type 2 Diabetes: Secondary Analyses of a Randomized Controlled Trial.

Food protein or food-derived peptides may regulate blood glucose levels; however, studies have shown inconsistent results. The aim of the present study was to characterize subgroups of individuals with increased risk of type 2 diabetes (T2D) and to investigate the cardiometabolic effects of fish protein in the same subgroups. We first divided participants into high insuliniAUC and low insuliniAUC subjects based on their insulin incremental area under the curve (iAUC) levels after a 2 h oral glucose tolerance test (OGTT), and secondly based on whether they had received 5.2 g salmon fish protein or placebo for 8 weeks, in a previously conducted randomized controlled trial (RCT). We then profiled these groups by analyzing plasma metabolomics and peripheral blood mononuclear cell (PBMC) gene expression. Compared to the low insuliniAUC group, the high insuliniAUC group had higher plasma concentrations of monounsaturated fatty acids (MUFAs) and glycated proteins (GlycA) and lower concentrations of glycine and acetate. After intervention with fish protein compared to placebo, however, only acetate was significantly increased in the low insuliniAUC group. In conclusion, we identified metabolic biomarkers known to be associated with T2D; also, intervention with fish protein did not affect cardiometabolic risk markers in subgroups with increased risk of T2D.

Consumption of salmon fishmeal increases hepatic cholesterol content in obese C57BL/6 J mice.

PURPOSE: By-products from farmed fish contain large amounts of proteins and may be used for human consumption. The purpose of this study was to investigate cardiometabolic effects and metabolic tolerance in mice consuming fishmeal from salmon by-products, salmon filet or beef. METHODS: Female C57BL/6J mice were fed chow, as a healthy reference group, or a high-fat diet for 10 weeks to induce obesity and glucose intolerance. Obese mice were subsequently given isocaloric diets containing 50% of the dietary protein from salmon fishmeal, salmon filet or beef for 10 weeks. Mice were subjected to metabolic phenotyping, which included measurements of body composition, energy metabolism in metabolic cages and glucose tolerance. Lipid content and markers of hepatic toxicity were determined in plasma and liver. Hepatic gene and protein expression was determined with RNA sequencing and immunoblotting. RESULTS: Mice fed fishmeal, salmon filet or beef had similar food intake, energy consumption, body weight gain, adiposity, glucose tolerance and circulating levels of lipids and hepatic toxicity markers, such as p-ALT and p-AST. Fishmeal increased hepatic cholesterol levels by 35-36% as compared to salmon filet (p = 0.0001) and beef (p = 0.005). This was accompanied by repressed expression of genes involved in steroid and cholesterol metabolism and reduced levels of circulating Pcsk9. CONCLUSION: Salmon fishmeal was well tolerated, but increased hepatic cholesterol content. The high cholesterol content in fishmeal may be responsible for the effects on hepatic cholesterol metabolism. Before introducing fishmeal from salmon by-products as a dietary component, it may be advantageous to reduce the cholesterol content in fishmeal.

Postprandial Effects of Salmon Fishmeal and Whey on Metabolic Markers in Serum and Gene Expression in Liver Cells.

Fish is considered an important part of a healthy diet, in part due to the content of long chain omega-3 fatty acids. However, both lean and fatty fish have beneficial health effects, suggesting that micronutrients and proteins may play a role. In a randomised, controlled, cross-over trial, five healthy male participants consumed 5.2 g of protein from either salmon fishmeal or whey. Blood samples were taken before and 30 and 60 min after intake. The concentration of glucose, lipids, hormones and metabolites, including 28 different amino acids and derivatives, were measured in serum or plasma. Cultured HepG2 cells were incubated with or without serum from the participants, and transcriptomic profiling was performed using RNA sequencing. The ingestion of both salmon fishmeal and whey reduced the glucose and triglyceride levels in serum. Protein intake, independent of the source, increased the concentration of 22 amino acids and derivatives in serum. Fishmeal increased the concentration of arginine, methionine, serine, glycine, cystathionine and 2-aminobutyric acid more than whey did. Incubation with postprandial serum resulted in large transcriptomic alterations in serum-fasted HepG2 cells, with the differential expression of >4500 protein coding genes. However, when comparing cells cultivated in fasting serum to postprandial serum after the ingestion of fishmeal and whey, we did not detect any differentially regulated genes, neither with respect to the protein source nor with respect to the time after the meal. The comparable nutrigenomic effects of fishmeal and whey do not change the relevance of fish by-products as an alternative food source.

Salmon fish protein supplement increases serum vitamin B12 and selenium concentrations: secondary analysis of a randomised controlled trial.

PURPOSE: The main aim of the present study was to examine the effect of a fish protein supplement made from by-products from production of Atlantic salmon, on blood concentration of micronutrients. METHODS: We conducted an 8-week double-blind parallel-group randomised controlled trial. In total, 88 adults were randomised to a salmon fish protein supplement or placebo, and 74 participants were included in the analysis of vitamin D, omega-3, vitamin B12, selenium, folate, zinc, homocysteine and mercury. RESULTS: During the intervention period, geometric mean (GSD) of serum vitamin B12 concentrations increased from 304 (1.40) to 359 (1.42) pmol/L in the fish protein group (P vs. controls = 0.004) and mean (SD) serum selenium increased from 1.18 (0.22) to 1.30 (0.20) µmol/L (P vs. controls = 0.002). The prevalence of low vitamin B12 status (B12 < 148-221 > pmol/L) decreased from 15.4 to 2.6% in the fish protein group, while increasing from 5.9 to 17.6% in the placebo group (P = 0.045). There was no difference between the groups in serum levels of the other micronutrients measured. CONCLUSION: Including a salmon fish protein supplement in the daily diet for 8 weeks, increases serum vitamin B12 and selenium concentrations. From a sustainability perspective, by-products with high contents of micronutrients and low contents of contaminants, could be a valuable dietary supplement or food ingredient in populations with suboptimal intake. TRAIL REGISTRATION: The study was registered at ClinicalTrials.gov (ID: NCT03764423) on June 29th 2018.

Methylation-dependent SUMOylation of the architectural transcription factor HMGA2.

High mobility group A2 (HMGA2) is a chromatin-associated protein involved in the regulation of stem cell function, embryogenesis and cancer development. Although the protein does not contain a consensus SUMOylation site, it is shown to be SUMOylated. In this study, we demonstrate that the first lysine residue in the reported K66KAE SUMOylation motif in HMGA2 can be methylated in vitro and in vivo by the Set7/9 methyltransferase. By editing the lysine, the increased hydrophobicity of the resulting 6-N-methyl-lysine transforms the sequence into a consensus SUMO motif. This post-translational editing dramatically increases the subsequent SUMOylation of this site. Furthermore, similar putative methylation-dependent SUMO motifs are found in a number of other chromatin factors, and we confirm methylation-dependent SUMOylation of a site in one such protein, the Polyhomeotic complex 1 homolog (PHC1). Together, these results suggest that crosstalk between methylation and SUMOylation is a general mode for regulation of chromatin function.

LXRα Regulates ChREBPα Transactivity in a Target Gene-Specific Manner through an Agonist-Modulated LBD-LID Interaction.

The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in the liver. More knowledge of their mechanistic interplay is needed to understand their role in pathological conditions like fatty liver disease and insulin resistance. In the current study, LXR and ChREBP co-occupancy was examined by analyzing ChIP-seq datasets from mice livers. LXR and ChREBP interaction was determined by Co-immunoprecipitation (CoIP) and their transactivity was assessed by real-time quantitative polymerase chain reaction (qPCR) of target genes and gene reporter assays. Chromatin binding capacity was determined by ChIP-qPCR assays. Our data show that LXRα and ChREBPα interact physically and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP but not for LXR, indicating that ChREBPα recruits LXRα to chromatin in trans. The two factors interact via their key activation domains; the low glucose inhibitory domain (LID) of ChREBPα and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly represses ChREBPα activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXRα:ChREBPα interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This ligand-driven molecular switch highlights an unappreciated role of LXRα in responding to nutritional cues that was overlooked due to LXR lipogenesis-promoting function.

Nuclear Lamin B1 Interactions With Chromatin During the Circadian Cycle Are Uncoupled From Periodic Gene Expression.

Many mammalian genes exhibit circadian expression patterns concordant with periodic binding of transcription factors, chromatin modifications, and chromosomal interactions. Here we investigate whether chromatin periodically associates with nuclear lamins. Entrainment of the circadian clock is accompanied, in mouse liver, by a net gain of lamin B1-chromatin interactions genome-wide, after which the majority of lamina-associated domains (LADs) are conserved during the circadian cycle. By tailoring a bioinformatics pipeline designed to identify periodic gene expression patterns, we also observe hundreds of variable lamin B1-chromatin interactions among which oscillations occur at 64 LADs, affecting one or both LAD extremities or entire LADs. Only a small subset of these oscillations however exhibit highly significant 12, 18, 24, or 30 h periodicity. These periodic LADs display oscillation asynchrony between their 5' and 3' borders, and are uncoupled from periodic gene expression within or in the vicinity of these LADs. Periodic gene expression is also unrelated to variations in gene-to-nearest LAD distances detected during the circadian cycle. Accordingly, periodic genes, including central clock-control genes, are located megabases away from LADs throughout circadian time, suggesting stable residence in a transcriptionally permissive chromatin environment. We conclude that periodic LADs are not a dominant feature of variable lamin B1-chromatin interactions during the circadian cycle in mouse liver. Our results also suggest that periodic hepatic gene expression is not regulated by rhythmic chromatin associations with the nuclear lamina.

Molecular modelling, synthesis, and biological evaluations of a 3,5-disubstituted isoxazole fatty acid analogue as a PPARα-selective agonist.

The peroxisome proliferator activated receptors (PPARs) are important drug targets in treatment of metabolic and inflammatory disorders. Fibrates, acting as PPARα agonists, have been widely used lipid-lowering agents for decades. However, the currently available PPARα targeting agents show low subtype-specificity and consequently a search for more potent agonists have emerged. In this study, previously isolated oxohexadecenoic acids from the marine algae Chaetoceros karianus were used to design a PPARα-specific analogue. Herein we report the design, synthesis, molecular modelling studies and biological evaluations of the novel 3,5-disubstituted isoxazole analogue 6-(5-heptyl-1,2-oxazol-3-yl)hexanoic acid (1), named ADAM. ADAM shows a clear receptor preference and significant dose-dependent activation of PPARα (EC50 = 47 µM) through its ligand-binding domain (LBD). Moreover, ADAM induces expression of important PPARα target genes, such as CPT1A, in the Huh7 cell line and primary mouse hepatocytes. In addition, ADAM exhibits a moderate ability to regulate PPARγ target genes and drive adipogenesis. Molecular modelling studies indicated that ADAM docks its carboxyl group into opposite ends of the PPARα and -γ LBD. ADAM interacts with the receptor-activating polar network of amino acids (Tyr501, His447 and Ser317) in PPARα, but not in PPARγ LBD. This may explain the lack of PPARγ agonism, and argues for a PPARα-dependent adipogenic function. Such compounds are of interest towards developing new lipid-lowering remedies.

Synthesis and biological evaluations of marine oxohexadecenoic acids: PPARα/γ dual agonism and anti-diabetic target gene effects.

Obesity and associated disorders such as metabolic syndrome and type 2 diabetes (T2D) have reached epidemic proportions. Several natural products have been reported as Peroxisome Proliferator-Activated Receptor (PPAR) agonists, functioning as lead compounds towards developing new anti-diabetic drugs due to adverse side effects of existing PPAR drugs. We recently isolated and identified (7E)-9-oxohexadec-7-enoic acid (1) and (10E)-9-oxohexadec-10-enoic acid (2) from the marine algae Chaetoceros karianus. Herein we report the total synthesis, pharmacological characterization, and biological evaluations of these naturally occurring oxo-fatty acids (oFAs). The syntheses of 1 and 2 afforded sufficient material for extensive biological evaluations. Both oFAs show an appreciable dose-dependent activation of PPARα and -γ, with EC50 values in the micromolar range, and an ability to regulate important PPAR target genes in hepatocytes and adipocytes. Moreover, both 1 and 2 are able to drive adipogenesis when evaluated in the Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell model, but with lowered expression of adipocyte markers and reduced lipid accumulation compared to the drug rosiglitazone. This seems to be caused by a transient upregulation of PPARγ and C/EBPα expression. Importantly, whole transcriptome analysis shows that both compounds induce anti-diabetic gene programs in adipocytes by upregulating insulin-sensitizing adipokines and repressing pro-inflammatory cytokines.

O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.

The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked ß-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20LWKPGAQDASSQAQGGSSCILRE42. However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT.

Two Isomeric C16 Oxo-Fatty Acids from the Diatom Chaetoceros karianus Show Dual Agonist Activity towards Human Peroxisome Proliferator-Activated Receptors (PPARs) α/γ.

The peroxisome proliferator-activated receptors (PPARs) function as ligand-activated transcription factors that convert signals in the form of lipids to physiological responses through the activation of metabolic target genes. Due to their key roles in lipid and carbohydrate metabolism, the PPARs are important drug targets. However, for several of the PPAR drugs currently in use, adverse side effects have been reported. In an effort to identify compounds from marine organisms that may serve as molecular scaffolds for the development of novel and safer PPAR-targeting drugs, we performed a bioassay-guided screening of organic extracts made from organisms supplied by the Norwegian Biobank of Arctic Marine Organisms (Marbank). Among several interesting hits, we identified two poorly described isomeric oxo-fatty acids from the microalgae Chaetoceros karianus for which we provide the first evidence that they might display dual specificity towards human PPARα and PPARγ. Principal component analysis showed that C. karianus stood out from other Chaetoceros species, both with respect to the metabolic profile and the PPAR activity. The isolation of these compounds holds the potential of uncovering a PPAR pharmacophore with tunable activity and specificity.

Differences in serum levels of CB-153 and p,p'-DDE, and reproductive parameters between men living south and north in Norway.

Arctic is contaminated with persistent organochlorine pollutants (POPs), and exposure to these compounds may differ between south and north in Norway. POPs may have negative impact on male reproductive characteristics. We compared serum levels of the CB-153 and p,p'-DDE in men who were born and had lived most of their lifetime south and north (close to or above the Arctic Circle) in Norway. We found no geographical differences in levels of CB-153 (south: 50 ng/g lipid (mean), north: 59 ng/g lipid; p=0.27) or sperm parameters. However, the levels of p,p'-DDE were higher in south than in north (81 ng/g lipid (mean) vs. 66 ng/g lipid; p=0.02), as were the levels of total and free testosterone. The FSH levels were lowest in south. A strong relationship between the CB-153 and the SHBG levels was observed. The regional differences observed for p,p'-DDE, testosterone and FSH were not reflected in the semen quality.

PIAS1 interacts with FLASH and enhances its co-activation of c-Myb.

BACKGROUND: FLASH is a huge nuclear protein involved in various cellular functions such as apoptosis signalling, NF-κB activation, S-phase regulation, processing of histone pre-mRNAs, and co-regulation of transcription. Recently, we identified FLASH as a co-activator of the transcription factor c-Myb and found FLASH to be tightly associated with active transcription foci. As a huge multifunctional protein, FLASH is expected to have many interaction partners, some which may shed light on its function as a transcriptional regulator. RESULTS: To find additional FLASH-associated proteins, we performed a yeast two-hybrid (Y2H) screening with FLASH as bait and identified the SUMO E3 ligase PIAS1 as an interaction partner. The association appears to involve two distinct interaction surfaces in FLASH. We verified the interaction by Y2H-mating, GST pulldowns, co-IP and ChIP. FLASH and PIAS1 were found to co-localize in nuclear speckles. Functional assays revealed that PIAS1 enhances the intrinsic transcriptional activity of FLASH in a RING finger-dependent manner. Furthermore, PIAS1 also augments the specific activity of c-Myb, and cooperates with FLASH to further co-activate c-Myb. The three proteins, FLASH, PIAS1, and c-Myb, are all co-localized with active RNA polymerase II foci, resembling transcription factories. CONCLUSIONS: We conclude that PIAS1 is a common partner for two cancer-related nuclear factors, c-Myb and FLASH. Our results point to a functional cooperation between FLASH and PIAS1 in the enhancement of c-Myb activity in active nuclear foci.

Identification of c-Myb Target Genes in K562 Cells Reveals a Role for c-Myb as a Master Regulator.

The c-Myb transcription factor is an important regulator of hematopoietic cell development. c-Myb is expressed in immature hematopoietic cells and plays a direct role in lineage fate selection, cell cycle progression, and differentiation of myeloid as well as B- and T-lymphoid progenitor cells. As a DNA-binding transcription factor, c-Myb regulates specific gene programs through activation of target genes. Still, our understanding of these programs is incomplete. Here, we report a set of novel c-Myb target genes, identified using a combined approach: specific c-Myb knockdown by 2 different siRNAs and subsequent global expression profiling, combined with the confirmation of direct binding of c-Myb to the target promoters by ChIP assays. The combination of these 2 approaches, as well as additional validation such as cloning and testing the promoters in reporter assays, confirmed that MYADM, LMO2, GATA2, STAT5A, and IKZF1 are target genes of c-Myb. Additional studies, using chromosome conformation capture, demonstrated that c-Myb target genes may directly interact with each other, indicating that these genes may be coordinately regulated. Of the 5 novel target genes identified, 3 are transcription factors, and one is a transcriptional co-regulator, supporting a role of c-Myb as a master regulator controlling the expression of other transcriptional regulators in the hematopoietic system.

A SUMO-regulated activation function controls synergy of c-Myb through a repressor-activator switch leading to differential p300 recruitment.

Synergy between transcription factors operating together on complex promoters is a key aspect of gene activation. The ability of specific factors to synergize is restricted by sumoylation (synergy control, SC). Focusing on the haematopoietic transcription factor c-Myb, we found evidence for a strong SC linked to SUMO-conjugation in its negative regulatory domain (NRD), while AMV v-Myb has escaped this control. Mechanistic studies revealed a SUMO-dependent switch in the function of NRD. When NRD is sumoylated, the activity of c-Myb is reduced. When sumoylation is abolished, NRD switches into being activating, providing the factor with a second activation function (AF). Thus, c-Myb harbours two AFs, one that is constitutively active and one in the NRD being SUMO-regulated (SRAF). This double AF augments c-Myb synergy at compound natural promoters. A similar SUMO-dependent switch was observed in the regulatory domains of Sp3 and p53. We show that the change in synergy behaviour correlates with a SUMO-dependent differential recruitment of p300 and a corresponding local change in histone H3 and H4 acetylation. We therefore propose a general model for SUMO-mediated SC, where SUMO controls synergy by determining the number and strength of AFs associated with a promoter leading to differential chromatin signatures.

Single semen analysis as a predictor of semen quality: clinical and epidemiological implications.

It is generally thought that a single ejaculate is a bad predictor of semen quality of a subject, because of significant intra-individual variation. Therefore, we investigated the degree to which the results of a first semen analysis differ from that of a second analysis among men from a general population in Norway. In addition, we analysed how the two different semen results mirrored the overall semen quality assessment. A total of 199 volunteers participated in the study and delivered two semen samples with an interval of 6 months. The semen parameters were determined according to the World Health Organization (WHO) 1999 guidelines, which were also used to determine whether semen quality was normal or abnormal. In addition, the DNA fragmentation index (DFI) was determined using the Sperm Chromatin Structure Assay. The two samples from each individual were very similar with regard to standard semen parameters and DFI (r(s:) 0.67-0.72), and there were no significant systematic differences between the two samples. The result of the first sample (normal/abnormal) was highly predictive of the overall conclusion based on the two samples (sperm concentration: in 93% of the cases (95% confidence interval [CI]: 89%-96%); sperm motility: in 85% of the cases (95% CI: 79%-89%); overall semen quality: in 85% of the cases (95% CI: 80%-90%). In epidemiological studies, one ejaculate is a sufficient indicator of semen quality in a group of subjects. In a clinical situation, when the question is whether the semen quality is normal or not, the first ejaculate will, in at least 85% of cases, give a correct overall conclusion.

Revisiting a selection of target genes for the hematopoietic transcription factor c-Myb using chromatin immunoprecipitation and c-Myb knockdown.

The transcription factor c-Myb is an important regulator of hematopoiesis required for proper development of most blood cell lineages in vertebrates. An increasing number of target genes for c-Myb are being published, although with little or no overlap between the lists of genes reported. This raises the question of which criteria a bona fide c-Myb-target gene should satisfy. In the present paper, we have analyzed a set of previously reported target genes using chromatin immunoprecipitation (ChIP) and siRNA-mediated knockdown. Among the seven well-studied c-Myb target genes that we analyzed by ChIP, only ADA, c-MYC and MAT2A seemed to be occupied by c-Myb under our experimental settings in the Myb-positive cell lines Jurkat and HL60. After siRNA-mediated knockdown of c-Myb expression, the expression levels of two out of three ChIP positive Myb target genes, ADA and c-MYC, were strongly affected. These results clearly demonstrate the importance of combining different methods for target gene validation and suggest that a combination of ChIP and c-Myb knockdown may represent a powerful approach to identify a core collection of c-Myb target genes.

Seasonal variation in serum concentrations of reproductive hormones and urinary excretion of 6-sulfatoxymelatonin in men living north and south of the Arctic Circle: a longitudinal study.

OBJECTIVE: Seasonal variation in photoperiod or temperature may influence human reproductive biology. The present study evaluated whether seasonal changes occurred in the levels of reproductive hormones and the major melatonin metabolite, 6-sulfatoxymelatonin (aMT6s), in populations exposed to extreme variation in photoperiod and temperature. DESIGN: Two separate cohorts of Norwegian men were recruited from the general population in either of two locations: Tromsø (69.5 degrees N, n = 92) or Oslo (60 degrees N, n = 112), located north and south of the Arctic Circle (66.5 degrees N), respectively. MEASUREMENTS: Four blood and 12-h overnight urine samples were obtained on separate occasions over a 12-month period, including during the photoperiod maximum and minimum. Serum concentrations of FSH, LH, testosterone (T), oestradiol (E(2)), SHBG and the urinary excretion of aMT6s were assessed. RESULTS: Statistical analysis using generalized estimating equations indicated that LH levels were lowest during early winter in both locations (both P = 0.01). In Tromsø, free T and E(2) concentrations peaked during early winter (P = 0.02 and 0.003, respectively). In Oslo, free T levels were lowest during early winter (P = 0.06) whereas E(2) levels were lowest during late summer (P < 0.001). Urinary aMT6s concentrations were lowest during early summer in Tromsø and Oslo. Concentrations peaked during early winter in Tromsø (P < 0.001) and during late winter in Oslo (P < 0.001). CONCLUSIONS: LH levels exhibited similar changes in both locations, whereas the patterns of changes of the sex steroid concentrations differed, possibly indicating different underlying mechanisms. Excretion of aMT6s was lowest during early summer in both locations, indicating that the long natural photoperiod was sufficient to cause suppression of melatonin secretion. Whether these changes have any biological significance remains uncertain.

The chromatin remodeling factor Mi-2alpha acts as a novel co-activator for human c-Myb.

The c-Myb protein belongs to a group of early hematopoietic transcription factors that are important for progenitor generation and proliferation. These factors have been hypothesized to participate in establishing chromatin patterns specific for hematopoietic genes. In a two-hybrid screening we identified the chromatin remodeling factor Mi-2alpha as an interaction partner for human c-Myb. The main interacting domains were mapped to the N-terminal region of Mi-2alpha and the DNA-binding domain of c-Myb. Surprisingly, functional analysis revealed that Mi-2alpha, previously studied as a subunit in the NuRD co-repressor complex, enhanced c-Myb-dependent reporter activation. Consistently, knock-down of endogenous Mi-2alpha in c-Myb-expressing K562 cells, led to down-regulation of the c-Myb target genes NMU and ADA. When wild-type and helicase-dead Mi-2alpha were compared, the Myb-Mi-2alpha co-activation appeared to be independent of the ATPase/DNA helicase activity of Mi-2alpha. The rationale for the unexpected co-activator function seems to lie in a dual function of Mi-2alpha, by which this factor is able to repress transcription in a helicase-dependent and activate in a helicase-independent fashion, as revealed by Gal4-tethering experiments. Interestingly, desumoylation of c-Myb potentiated the Myb-Mi-2alpha transactivational co-operation, as did co-transfection with p300.