Suppressing Hepatic UGT1A1 Increases Plasma Bilirubin, Lowers Plasma Urobilin, Reorganizes Kinase Signaling Pathways and Lipid Species and Improves Fatty Liver Disease.

Several population studies have observed lower serum bilirubin levels in patients with non-alcoholic fatty liver disease (NAFLD). Yet, treatments to target this metabolic phenotype have not been explored. Therefore, we designed an N-Acetylgalactosamine (GalNAc) labeled RNAi to target the enzyme that clears bilirubin from the blood, the UGT1A1 glucuronyl enzyme (GNUR). In this study, male C57BL/6J mice were fed a high-fat diet (HFD, 60%) for 30 weeks to induce NAFLD and were treated subcutaneously with GNUR or sham (CTRL) once weekly for six weeks while continuing the HFD. The results show that GNUR treatments significantly raised plasma bilirubin levels and reduced plasma levels of the bilirubin catabolized product, urobilin. We show that GNUR decreased liver fat content and ceramide production via lipidomics and lowered fasting blood glucose and insulin levels. We performed extensive kinase activity analyses using our PamGene PamStation kinome technology and found a reorganization of the kinase pathways and a significant decrease in inflammatory mediators with GNUR versus CTRL treatments. These results demonstrate that GNUR increases plasma bilirubin and reduces plasma urobilin, reducing NAFLD and inflammation and improving overall liver health. These data indicate that UGT1A1 antagonism might serve as a treatment for NAFLD and may improve obesity-associated comorbidities.

Metabolite quantification: A fluorescence-based method for urine sample normalization prior to 1H-NMR analysis.

INTRODUCTION: Metabolomics is a multi-discipline approach to systems biology that provides a snapshot of the metabolic status of a cell, tissue, or organism. Metabolomics uses mass spectroscopy (MS) and nuclear magnetic resonance (NMR) to analyze biological samples for low molecular weight metabolites. OBJECTIVE: Normalize urine sample pre-acquisition to perform a targeted quantitative analysis of selected metabolites in rat urine. METHODS: Urine samples were provided from rats on a control diet (n = 10) and moderate sucrose diet (n = 8) collected in a metabolic cage during an eight hour fast. Urine from each sample was prepared by two different methods. One sample was a non-normalized sample of 1200 µL and the second sample was a variable volume-normalized to the concentration of urobilin in a standard sample of urine. The urobilin concentration in all samples was determined by fluorescence. Ten metabolites for each non-normalized and normalized urine sample were quantified by integration to an internal standard of DSS. RESULTS: Both groups showed an improvement in pH range going from non-normalized to normalized samples. In the group on the control diet, eight metabolites had significant improvement in range, while the remaining two metabolites had insignificant improvement in range comparing the non-normalized sample to the normalized sample. In the group on the moderate sucrose diet all ten metabolites showed significant improvement in range going from non-normalized to normalized samples. CONCLUSIONS: These findings describe a pre-acquisition method of urine normalization to adjust for differences in hydration state of each organism. This results in a narrower concentration range in a targeted analysis.

Rapid and sensitive naked eye detection of faecal pigments using their enhanced solid-state green fluorescence on a zinc acetate substrate.

The identification of trace faecal pigments in real-time and on-site detection remains a challenge for water quality monitoring. Herein, a simple, low-cost and rapid fluorescence-based analytical method has been developed in a solid matrix for faecal pigments like stercobilin and urobilin detection. This was made possible due to significant enhancement of green solid-state fluorescence (520 nm) by zinc(II) complexation with faecal pigments embedded in the surface of zinc acetate crystals. It enables naked-eye detection of these pigments even at a 10 µM level when excited with 365 nm blue-UV. It was demonstrated that easily available white cellulose paper strips or TLC silica plates coated with zinc acetate can be used as substrates. A photophysical study of solid-state faecal pigments-zinc(II) complexes suggests that green fluorescence enhancement results from the complexation, which can be attributed to the substantial decrease of the non-radiative decay rate (knr) as well as more efficient use of excitation light. The observation of reduced interference of humic acid fluorescence makes faecal pigment detection more efficient by this proposed method.

Photophysics of faecal pigments stercobilin and urobilin in aliphatic alcohols: introduction of a sensitive method for their detection using solvent phase extraction and fluorometry.

Faecal pigments (FPs) are ubiquitous in the environment and are a primary contaminant in groundwater and surface water. This article presents a new analytical paradigm by a fluorescence coupled extraction-based method involving FP fluorescence enhancement and minimization of background fluorescence for high sensitivity detection. FPs show higher fluorescence intensity in aliphatic alcohols due to the breaking down of higher-order H-aggregates into lower-order H-aggregates (dimers). DFT studies using the B3LYP functional and LANL2DZ basis set show π-π stacking and hydrogen-bonding contributions towards forming H-aggregated dimers of FPs in the implicit and explicit solvent environments of 1-hexanol. This study is the first report on the extractability of FPs using 1-hexanol as an efficient extraction medium in comparison to higher-order aliphatic alcohols (1-butanol, 1-hexanol and 1-octanol). Furthermore, FP-Zn(II) complexes in 1-hexanol medium significantly enhance the fluorescence emission intensity (∼14-17 times), and the emission intensity remains stable over time. This further helps to increase the detection limit of FPs in the picomolar to sub-picomolar concentration range. This study proposes a protocol involving extraction of FPs by 1-hexanol followed by the complexation of FPs with Zn(II) in the alcohol media and subsequent fluorimetric detection of the FP-Zn(II) complex with a high level of sensitivity, enabled by reduced interference from the background fluorescence of humic acid. The complexation behaviour of FPs with various metal salts was also examined, which provided an understanding of the fluorescence behaviour of FPs with various other metal ions commonly present in natural environmental water. The proposed analytical method has been further validated using real water samples.

Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria.

Marine Synechococcus cyanobacteria owe their ubiquity in part to the wide pigment diversity of their light-harvesting complexes. In open ocean waters, cells predominantly possess sophisticated antennae with rods composed of phycocyanin and two types of phycoerythrins (PEI and PEII). Some strains are specialized for harvesting either green or blue light, while others can dynamically modify their light absorption spectrum to match the dominant ambient color. This process, called type IV chromatic acclimation (CA4), has been linked to the presence of a small genomic island occurring in two configurations (CA4-A and CA4-B). While the CA4-A process has been partially characterized, the CA4-B process has remained an enigma. Here we characterize the function of two members of the phycobilin lyase E/F clan, MpeW and MpeQ, in Synechococcus sp. strain A15-62 and demonstrate their critical role in CA4-B. While MpeW, encoded in the CA4-B island and up-regulated in green light, attaches the green light-absorbing chromophore phycoerythrobilin to cysteine-83 of the PEII α-subunit in green light, MpeQ binds phycoerythrobilin and isomerizes it into the blue light-absorbing phycourobilin at the same site in blue light, reversing the relationship of MpeZ and MpeY in the CA4-A strain RS9916. Our data thus reveal key molecular differences between the two types of chromatic acclimaters, both highly abundant but occupying distinct complementary ecological niches in the ocean. They also support an evolutionary scenario whereby CA4-B island acquisition allowed former blue light specialists to become chromatic acclimaters, while former green light specialists would have acquired this capacity by gaining a CA4-A island.

Understanding the photophysics of stercobilin-Zn(II) and urobilin-Zn(II) complexes towards faecal pigment analysis.

A detailed photophysical study of two faecal pigments (FPs), Urobilin (UB) and Stercobilin (SB), and their zinc complexes [FP-Zn(II)] was carried out. The enhancement of UB and SB fluorescence resulting from the formation of their Zn(II) complexes was attributed to the complexation-induced rigidity of the chromophoric units, and the corresponding decrease of nonradiative decay rate constants of the excited singlet states (knr). The effect of various physicochemical environments was also studied in detail in order to understand the fluorescence behaviour of the Zn(II) complexes. FP-Zn(II) complexes have a lower solubility in water that results in the formation of molecular aggregates. The aggregation-induced loss of fluorescence of FP-Zn(II) complexes could be overcome by using the appropriate mixture of ethanol and water (70:30). Molecular orbital calculations on the FP-Zn(II) complexes provided a good idea of the geometry of the complexes and helped rationalise the enhancement of fluorescence after complexation. This study could pave the way towards developing a convenient non-extraction aqueous phase analytical procedure for detection of FPs using Zn(II) complexation method.

MpeV is a lyase isomerase that ligates a doubly linked phycourobilin on the ß-subunit of phycoerythrin I and II in marine Synechococcus.

Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the ß-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into ß-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases.

Biosynthesis of a Phycocyanin Beta Subunit with Two Noncognate Chromophores in Escherichia coli.

Recombinant phycobiliprotein can be used as fluorescent label in immunofluorescence assay. In this study, pathway for phycocyanin beta subunit (CpcB) carrying noncognate chromophore phycoerythrobilin (PEB) and phycourobilin (PUB) was constructed in Escherichia coli. Lyase CpcS and CpcT could catalyze attachment of PEB to Cys84-CpcB and Cys155-CpcB, respectively. However, PEB was attached only to Cys84-CpcB when both CpcS and CpcT were present in E. coli. A dual plasmid expression system was used to control the expression of lyases and the attachment order of PEB to CpcB. The production of PEB-Cys155-CpcB was achieved by L-arabinose-induced expression of CpcS, CpcB, Ho1, and PebS, and then the attachment of PEB to Cys84-CpcB was achieved by IPTG-induced expression of CpcS. The doubly chromophorylated CpcB absorbed light maximally at 497.5 nm and 557.0 nm and fluoresced maximally at 507.5 nm and 566.5 nm. An amount of light energy absorbed by PUB-Cys155-CpcB is transferred to PEB-Cys84-CpcB in doubly chromophorylated CpcB, conferring a large stokes shift of 69 nm for this fluorescent protein. There are interactions between chromophores of CpcB which possibly together with the help of lyases lead to isomerization of PEB-Cys155-CpcB to PUB-Cys155-CpcB.

Evaluating Novel Markers for Specimen Validity Testing.

CONTEXT.­: Synthetic urine products are commercially marketed for the purpose of specimen substitution for urine drug screens. These products are widely popular because they yield negative drug screen results, meet criteria for specimen validity testing, and are easily accessible and affordable. Current specimen validity criteria are ineffective for detecting these synthetic products, and new markers of specimen validity are required. OBJECTIVE.­: To develop and evaluate a multicomponent liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for urine specimen validity testing. DESIGN.­: A quantitative LC-MS/MS assay was developed for caffeine, cotinine, theobromine, and urobilin in urine. The assay was applied to known synthetic urine products (n = 10) as well as human specimens received for pre-employment testing (n = 500), for-cause workplace testing (n = 100), and medical pain management monitoring (n = 200). Specimens devoid of all 4 validity markers were subjected to follow-up testing that involved microscopic urinalysis and comprehensive gas chromatography mass spectrometry for drugs, pharmaceuticals, hormones, and lipids. RESULTS.­: Of the experimental groups, 10 of 10 synthetic urine products (100%), 12 of 500 pre-employment specimens (2.4%), and 4 of 200 pain management specimens (2.0%) failed the experimental LC-MS/MS assay. Follow-up testing indicated that each of the failed specimens was nonphysiologic in nature. CONCLUSIONS.­: Simultaneous application of the 4 experimental validity markers appeared to be a robust method for detecting nonphysiologic specimens. New markers of specimen validity must be developed in order to identify commercially available synthetic urine products.

The metabolites urobilin and sphingomyelin (30:1) are associated with incident heart failure in the general population.

AIMS: We aimed to investigate whether metabolomic profiling of blood can lead to novel insights into heart failure pathogenesis or improved risk prediction. METHODS AND RESULTS: Mass spectrometry-based metabolomic profiling was performed in plasma or serum samples from three community-based cohorts without heart failure at baseline (total n = 3924; 341 incident heart failure events; median follow-up ranging from 4.6 to 13.9 years). Cox proportional hazard models were applied to assess the association of each of the 206 identified metabolites with incident heart failure in the discovery cohorts Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) (n = 920) and Uppsala Longitudinal Study of Adult Men (ULSAM) (n = 1121). Replication was undertaken in the independent cohort TwinGene (n = 1797). We also assessed whether metabolites could improve the prediction of heart failure beyond established risk factors (age, sex, body mass index, low-density and high-density lipoprotein cholesterol, triglycerides, lipid medication, diabetes, systolic and diastolic blood pressure, blood pressure medication, glomerular filtration rate, smoking status, and myocardial infarction prior to or during follow-up). Higher circulating urobilin and lower sphingomyelin (30:1) were associated with incident heart failure in age-adjusted and sex-adjusted models in the discovery and replication sample. The hazard ratio for urobilin in the replication cohort was estimated to 1.29 per standard deviation unit, 95% confidence interval (CI 1.03-1.63), and for sphingomyelin (30:1) to 0.72 (95% CI 0.58-0.89). Results remained similar after further adjustment for established heart failure risk factors in meta-analyses of all three cohorts. Urobilin concentrations were inversely associated with left ventricular ejection fraction at baseline in the PIVUS cohort (ß = -0.70, 95% CI -1.03 to -0.38). No major improvement in risk prediction was observed when adding the top 2 metabolites (C-index 0.787, 95% CI 0.752-0.823) or nine Lasso-selected metabolites (0.790, 95% CI 0.754-0.826) to a modified Atherosclerosis Risk in Communities heart failure risk score model (0.780, 95% CI 0.745-0.816). CONCLUSIONS: Our metabolomic profiling of three community-based cohorts study identified associations of circulating levels of the haem breakdown product urobilin, and sphingomyelin (30:1), a cell membrane component involved in signal transduction and apoptosis, with incident heart failure.

Interplay between differentially expressed enzymes contributes to light color acclimation in marine Synechococcus.

Marine Synechococcus, a globally important group of cyanobacteria, thrives in various light niches in part due to its varied photosynthetic light-harvesting pigments. Many Synechococcus strains use a process known as chromatic acclimation to optimize the ratio of two chromophores, green-light-absorbing phycoerythrobilin (PEB) and blue-light-absorbing phycourobilin (PUB), within their light-harvesting complexes. A full mechanistic understanding of how Synechococcus cells tune their PEB to PUB ratio during chromatic acclimation has not yet been obtained. Here, we show that interplay between two enzymes named MpeY and MpeZ controls differential PEB and PUB covalent attachment to the same cysteine residue. MpeY attaches PEB to the light-harvesting protein MpeA in green light, while MpeZ attaches PUB to MpeA in blue light. We demonstrate that the ratio of mpeY to mpeZ mRNA determines if PEB or PUB is attached. Additionally, strains encoding only MpeY or MpeZ do not acclimate. Examination of strains of Synechococcus isolated from across the globe indicates that the interplay between MpeY and MpeZ uncovered here is a critical feature of chromatic acclimation for marine Synechococcus worldwide.

The γ33 subunit of R-phycoerythrin from Gracilaria chilensis has a typical double linked phycourobilin similar to ß subunit.

Phycobilisomes (PBS) are accessory light harvesting protein complexes formed mainly by phycobiliproteins (PBPs). The PBPs absorb light that is efficiently transferred to Photosystems due to chromophores covalently bound to specific cysteine residues. Besides phycobiliproteins (PE), the PBS contains linker proteins responsible for assembly and stabilization of the whole complex and the tuning of energy transfer steps between chromophores. The linker (γ33) from Gracilaria chilensis, is a chromophorylated rod linker associated to (αß)6 hexamers of R-phycoerythrin (R-PE). Its role in the energy transfer process is not clear yet. Structural studies as well as the composition and location of the chromophores are essential to understand their involvement in the energy transfer process in PBS. To achieve this, the coding gene of γ33 was cloned and sequenced. The sequence was analyzed by informatics tools, to obtain preliminary information which leaded the next experiments. The protein was purified from R-phycoerythrin, and the sequence confirmed by mass spectrometry. The coding sequence analysis revealed a protein of 318 aminoacid residues containing a chloroplastidial transit peptide (cTP) of 39 aminoacids at the N-terminus. The conservation of cysteines revealed possible chromophorylation sites. Using α and ß R-PE subunits as spectroscopic probes in denaturation assays, we deduced a double bonded phycourobilin (PUB) on γ33 subunit that were confirmed between Cys62 and Cys73 (DL-PUB62/73) by mass spectrometry. The cysteines involved in the double link are located in a helical region, in a conformation that reminds the position of the DL-PUB50/61 in the ß subunit of R-PE. The position of single linked PUB at Cys95 and a single linked PEB at Cys172 were also confirmed. Spectroscopic studies show the presence of both types of chromophores and that there are not energy transfer by FRET among them.

Chemical Assay for the Detection of Vertebrate Fecal Metabolites in Adult Blow Flies (Diptera: Calliphoridae).

Filth flies are commonly implicated in pathogen transmission routes due to their affinity for vertebrate waste and their synanthropic associations. However, solidifying the link between flies and infected feces in the wild can be difficult, as interpretations made solely from microbial culturing or sequencing methods may represent an incomplete picture of pathogen acquisition. We present an analytical assay using high performance liquid chromatography tandem mass spectrometry (HPLC MS/MS) to detect vertebrate fecal metabolites (urobilinoids) in adult blow fly guts. Proof of concept experiments consisted of controlled feeding in which flies were grouped into three treatments (unfed, exposure to beef liver tissue, and exposure to canine feces; N = 20/treatment) using the black blow fly Phormia regina Meigen (Diptera: Calliphoridae). It was revealed that only feces-related samples exhibited peaks with an m/z of 591 and MS/MS spectra consistent with urobilinoids. These peaks were not seen for beef liver tissue, flies exposed to beef liver tissue, or unfed flies. Samples taken directly from beef liver tissue and from feces of several animals were also tested. To test this assay in wild flies, 216 flies were additionally analyzed to determine whether they had ingested vertebrate feces. About 13% of the wild flies exhibited these same peaks, providing a baseline measure of blow flies collected in urban and residential areas consuming feces from the environment. Overall, this assay can be used for P. regina collected in an applied setting and its integration with microbial culturing and sequencing methods will help to improve its use.

Physiological and proteomic characterization of light adaptations in marine Synechococcus.

Marine Synechococcus thrive over a range of light regimes in the ocean. We examined the proteomic, genomic and physiological responses of seven Synechococcus isolates to moderate irradiances (5-80 µE m-2 s-1 ), and show that Synechococcus spans a continuum of light responses ranging from low light optimized (LLO) to high light optimized (HLO). These light responses are linked to phylogeny and pigmentation. Marine sub-cluster 5.1A isolates with higher phycouribilin: phycoerythrobilin ratios fell toward the LLO end of the continuum, while sub-cluster 5.1B, 5.2 and estuarine Synechococcus with less phycouribilin fell toward the HLO end of the continuum. Global proteomes were highly responsive to light, with > 50% of abundant proteins varying more than twofold between the lowest and highest irradiance. All strains downregulated phycobilisome proteins with increasing irradiance. Regulation of proteins involved in photosynthetic electron transport, carbon fixation, oxidative stress protection (superoxide dismutases) and iron and nitrogen metabolism varied among strains, as did the number of high light inducible protein (Hlip) and DNA photolyase genes in their genomes. All but one LLO strain possessed the photoprotective orange carotenoid protein (OCP). The unique combinations of light responses in each strain gives rise to distinct photophysiological phenotypes that may affect Synechococcus distributions in the ocean.

Phylogeography and pigment type diversity of Synechococcus cyanobacteria in surface waters of the northwestern pacific ocean.

The widespread unicellular cyanobacteria Synechococcus are major contributors to global marine primary production. Here, we report their abundance, phylogenetic diversity (as assessed using the RNA polymerase gamma subunit gene rpoC1) and pigment diversity (as indirectly assessed using the laterally transferred cpeBA genes, encoding phycoerythrin-I) in surface waters of the northwestern Pacific Ocean, sampled over nine distinct cruises (2008-2015). Abundance of Synechococcus was low in the subarctic ocean and South China Sea, intermediate in the western subtropical Pacific Ocean, and the highest in the Japan and East China seas. Clades I and II were by far the most abundant Synechococcus lineages, the former dominating in temperate cold waters and the latter in (sub)tropical waters. Clades III and VI were also fairly abundant in warm waters, but with a narrower distribution than clade II. One type of chromatic acclimater (3dA) largely dominated the Synechococcus communities in the subarctic ocean, while another (3dB) and/or cells with a fixed high phycourobilin to phycoerythrobilin ratio (pigment type 3c) predominated at mid and low latitudes. Altogether, our results suggest that the variety of pigment content found in most Synechococcus clades considerably extends the niches that they can colonize and therefore the whole genus habitat.

Genetic and ecophysiological traits of Synechococcus strains isolated from coastal and open ocean waters of the Arabian Sea.

The picocyanobacterium Synechococcus is a prominent primary producer in the marine environment. The marine Synechococcus strains are clustered into different clades representing ecologically distinct genotypes. In this study, we compared phylogeny, photophysiology and cell cycles of four novel phycoerythrin-containing Synechococcus strains (clade II of subcluster 5.1) isolated from different depths of the water column (surface and subsurface waters) in coastal and offshore regions of the eastern Arabian Sea. The surface water strains possessed a lesser number of thylakoid layers and had a higher zeaxanthin to chlorophyll a ratio than subsurface strains indicating possible influence of light intensity available at their niche. The DNA distribution pattern of the four strains was bimodal in optimal cellular physiology conditions with cell division restricted to the light period and synchronized with the light-dark cycle. The presence of phycourobilin or phycoerythrobilin and the ratio between these two chromophores in all four strains varied according to available spectral wavelength in situ This study indicates that the timing of cell division is conserved within these genotypically identical Synechococcus strains, despite their having different chromophore ratios. We conclude that the timing of cell division of the Synechococcus strains has a genetic basis rather than being determined by phenotypic characters, such as chromophore content and ratio.

Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy.

Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment.

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin.

Cyanobacteriochromes are a structurally and spectrally highly diverse class of phytochrome-related photosensory biliproteins. They contain one or more GAF domains that bind phycocyanobilin (PCB) autocatalytically; some of these proteins are also capable of further modifying PCB to phycoviolobilin or rubins. We tested the chromophorylation with the non-photochromic phycoerythrobilin (PEB) of 16 cyanobacteriochrome GAFs from Nostoc sp. PCC 7120, of Slr1393 from Synechocystis sp. PCC 6803, and of Tlr0911 from Thermosynechococcus elongatus BP-1. Nine GAFs could be autocatalytically chromophorylated in vivo/in E. coli with PEB, resulting in highly fluorescent biliproteins with brightness comparable to that of fluorescent proteins like GFP. In several GAFs, PEB was concomitantly converted to phycourobilin (PUB) during binding. This not only shifted the spectra, but also increased the Stokes shift. The chromophorylated GAFs could be oligomerized further by attaching a GCN4 leucine zipper domain, thereby enhancing the absorbance and fluorescence of the complexes. The presence of both PEB and PUB makes these oligomeric GAF-"bundles" interesting models for energy transfer akin to the antenna complexes found in cyanobacterial phycobilisomes. The thermal and photochemical stability and their strong brightness make these constructs promising orange fluorescent biomarkers.

The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay.

Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu(2+)) to cuprous ions (Cu(1+)), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.

In vitro DNA-damaging effects of intestinal and related tetrapyrroles in human cancer cells.

Epidemiological studies report a negative association between circulating bilirubin concentrations and the risk for cancer and cardiovascular disease. Structurally related tetrapyrroles also possess in vitro anti-genotoxic activity and may prevent mutation prior to malignancy. Furthermore, few data suggest that tetrapyrroles exert anti-carcinogenic effects via induction of cell cycle arrest and apoptosis. To further investigate whether tetrapyrroles provoke DNA-damage in human cancer cells, they were tested in the single cell gel electrophoresis assay (SCGE). Eight tetrapyrroles (unconjugated bilirubin, bilirubin ditaurate, biliverdin, biliverdin-/bilirubin dimethyl ester, urobilin, stercobilin and protoporphyrin) were added to cultured Caco2 and HepG2 cells and their effects on comet formation (% tail DNA) were assessed. Flow cytometric assessment (apoptosis/necrosis, cell cycle, intracellular radical species generation) assisted in revealing underlying mechanisms of intracellular action. Cells were incubated with tetrapyrroles at concentrations of 0.5, 5 and 17µM for 24h. Addition of 300µM tertiary-butyl hydroperoxide to cells served as a positive control. Tetrapyrrole incubation mostly resulted in increased DNA-damage (comet formation) in Caco2 and HepG2 cells. Tetrapyrroles that are concentrated within the intestine, including protoporphyrin, urobilin and stercobilin, led to significant comet formation in both cell lines, implicating the compounds in inducing DNA-damage and apoptosis in cancer cells found within organs of the digestive system.