The Cyanobacterial "Nutraceutical" Phycocyanobilin Inhibits Cysteine Protease Legumain.

The blue biliprotein phycocyanin, produced by photo-autotrophic cyanobacteria including spirulina (Arthrospira) and marketed as a natural food supplement or "nutraceutical," is reported to have anti-inflammatory, antioxidant, immunomodulatory, and anticancer activity. These diverse biological activities have been specifically attributed to the phycocyanin chromophore, phycocyanobilin (PCB). However, the mechanism of action of PCB and the molecular targets responsible for the beneficial properties of PCB are not well understood. We have developed a procedure to rapidly cleave the PCB pigment from phycocyanin by ethanolysis and then characterized it as an electrophilic natural product that interacts covalently with thiol nucleophiles but lacks any appreciable cytotoxicity or antibacterial activity against common pathogens and gut microbes. We then designed alkyne-bearing PCB probes for use in chemical proteomics target deconvolution studies. Target identification and validation revealed the cysteine protease legumain (also known as asparaginyl endopeptidase, AEP) to be a target of PCB. Inhibition of this target may account for PCB's diverse reported biological activities.

Assessment of the Anticancer Potentials of the Free and Metal-Organic Framework (UiO-66) - Delivered Phycocyanobilin.

Phycocyanin (C-PC) is a constitutive chromoprotein of Arthrospira platensis, which exhibits promising efficacy against different types of cancer. In this study, we cleaved C-PC's chromophore phycocyanobilin (PCB) and demonstrated its ability as an anti-cancer drug for Colorectal cancer (CRC). PCB displayed an anti-cancer effect for CRC (HT-29) cells with IC50 of 108 µg/ml. Assessing the transcripts levels of some biomarkers revealed that the PCB caused an upregulation in the anti-metastatic gene NME1 level and downregulation of the COX-2 level. The flow cytometric results showed the effect of PCB on the arrest of the cell cycle's G1 phase. In addition, we successfully synthesized the UiO-66 (Zr-MOF). We incorporated the PCB into UiO-66 nanoparticles with a loading percentage of 46 %. Assessment of the cytotoxic effects of UiO-66@PCB showed a 2-fold improvement in the IC50 compared to the free PCB. In conclusion, we have shown that PCB displayed a promising potential as an anti-cancer agent. Yet, it is considered a safe and natural substance that can help to mitigate cancer spread and symptoms. In the meantime, UiO-66 can be used as a safe nano-delivery tool for PCB.

Monitoring effect of nickel, copper, and zinc on growth and photosynthetic pigments of Spirulina platensis with suitability investigation in Idku Lake.

Owing to the increase of pollutant sources in oceans, seas, and lakes, there is an expected effect on growth and metabolism of planktonic algae which are considered primary producers in the ecosystem. Therefore, it becomes urgent to carry out laboratory studies to test to what extent these pollutants can affect the growth of algae which is necessary as a food for marine fishes. Spirulina is considered the most important algal species due to its high nutritional value for humans and animals. Therefore, this work investigated the effect of different concentrations of Ni2+, Zn2+, and Cu2+ metal ion pollutants on growth of the blue-green alga Spirulina platensis. EC50 was identified to be around 2 mg/l for the three heavy metals. The suitability of Idku Lake for Spirulina platensis growth was investigated using multi-criteria spatial modeling integrated with remotely sensed data processing. Spatial distribution maps of turbidity, water nutrients, and phytoplankton were the input criteria used to assess Idku Lake's suitability. The results obtained proved that low concentrations of the tested heavy metals stimulated growth and pigment fractions (chlorophyll a, carotenoids, and total phycobilins content) but to different degrees. The inhibitory effect was more prominent in the case of copper ions than zinc and nickel ions with all concentrations used. The overall suitability map of Spirulina platensis in Idku Lake showed that the whole lake is suitable for growth and proliferation except for the northwestern corner due to the high salinity levels. The present paper helps to understand the behavior of algae responding to environmental pollution, which supports environmental planners with the necessary baseline for investigating the fate of pollutants and the potential risk.

The Bioactivities of Phycocyanobilin from Spirulina.

Phycocyanobilin (PCB) is a linear open-chain tetrapyrrole chromophore that captures and senses light and a variety of biological activities, such as anti-oxidation, anti-cancer, and anti-inflammatory. In this paper, the biological activities of PCB are reviewed, and the related mechanism of PCB and its latest application in disease treatment are introduced. PCB can resist oxidation by scavenging free radicals, inhibiting the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and delaying the activity of antioxidant enzymes. In addition, PCB can also be used as an excellent anti-inflammatory agent to reduce the proinflammatory factors IL-6 and IFN-γ and to up-regulate the production of anti-inflammatory cytokine IL-10 by inhibiting the inflammatory signal pathways NF-κB and mitogen-activated protein kinase (MAPK). Due to the above biological activities of phycocyanobilin PCB, it is expected to become a new effective drug for treating various diseases, such as COVID-19 complications, atherosclerosis, multiple sclerosis (MS), and ischaemic stroke (IS).

Phycocyanin ameliorates mouse colitis via phycocyanobilin-dependent antioxidant and anti-inflammatory protection of the intestinal epithelial barrier.

Phycocyanin is a typical microalgal active compound with antioxidant and anti-inflammatory efficacy, and the pigment moiety phycocyanobilin has been recently proposed as its active structural component. Here, to explore the structural basis for phycocyanin's intestinal protective action, we evaluated the therapeutic effects and mechanism of action of phycocyanin and phycocyanobilin in dextran sodium sulphate (DSS)-induced colitis mice and in Caco-2 and RAW 264.7 cells. Phycocyanobilin was obtained by solvothermal alcoholysis of phycocyanin and characterized by spectroscopy and mass spectrometry methods. Phycocyanin, phycocyanobilin and a positive drug mesalazine were intragastrically administered to C57BL/6 mice daily for 7 days during and after 4-day DSS exposure. Clinical signs and colon histopathology revealed that phycocyanin and phycocyanobilin had an equivalent anti-colitis efficacy that was even superior to mesalazine. Based on biochemical analysis of colonic tight junction proteins, mucus compositions and goblet cells, and colonic and peripheral proinflammatory cytokines, phycocyanin and phycocyanobilin displayed equivalent intestinal epithelial barrier-protecting and anti-inflammatory potential that was evidently superior to that of mesalazine. Flow cytometry analysis of phycocyanobilin fluorescence in Caco-2 cells unveiled a similar uptake efficacy of phycocyanin and phycocyanobilin by intestinal epithelial cells. According to lactic dehydrogenase release, 2',7'-dichlorodihydrofluorescein fluorescence and methylthiazolyldiphenyl-tetrazolium bromide assay in Caco-2 cells, phycocyanin and phycocyanobilin could equally and effectively protect the intestinal epithelial barrier from oxidant-induced disruption. Phycocyanin and phycocyanobilin also showed equivalent anti-inflammatory effects in tumor necrosis factor-α-stimulated Caco-2 cells and in lipopolysaccharides- and tumor necrosis factor-α-activated RAW264.7 cells. Overall, our results demonstrate the phycocyanobilin-dependent anti-colitis role of phycocyanin via antioxidant and anti-inflammatory mechanisms.

Nutraceutical and therapeutic potential of Phycocyanobilin for treating Alzheimer's disease.

Alzheimer's disease (AD) is a devastating neurodegenerative condition provoking the loss of cognitive and memory performances. Despite huge efforts to develop effective AD therapies, there is still no cure for this neurological condition. Here, we review the main biological properties of Phycocyanobilin (PCB), accounting for its potential uses against AD. PCB, given individually or released in vivo from C-Phycocyanin (C-PC), acts as a bioactive-molecule-mediating antioxidant, is anti-inflammatory and has immunomodulatory activities. PCB/C-PC are able to scavenge reactive oxygen and nitrogen species, to counteract lipid peroxidation and to inhibit enzymes such as NADPH oxidase and COX-2. In animal models of multiple sclerosis and ischemic stroke, these compounds induce remyelination as demonstrated by electron microscopy and the expression of genes such as Mal up-regulation of and Lingo-1 down-regulation. These treatments also reduce pro-inflammatory cytokines levels and induce immune suppressive genes. PCB/C-PC protects isolated rat brain mitochondria and inactivate microglia, astrocytes and neuronal apoptosis mediators. Such processes are all involved in the pathogenic cascade of AD, and thus PCB may effectively mitigate the injury in this condition. Furthermore, PCB can be administered safely by oral or parenteral routes and therefore, could be commercially offered as a nutraceutical supplement or as a pharmaceutical drug.

Phycocyanobilin is the molecule responsible for the nephroprotective action of phycocyanin in acute kidney injury caused by mercury.

C-Phycocyanin (CPC) exerts therapeutic, antioxidant, anti-inflammatory and immunomodulatory actions. It prevents oxidative stress and acute kidney damage caused by HgCl2. However, the exact mechanism of the pharmacological action of C-phycocyanin is as yet unclear. Some proposals express that CPC metabolism releases the active compound phycocyanobilin (PCB) that is able to induce CPC's therapeutical effects as an antioxidant, anti-inflammatory and nephroprotective. This study is aimed to demonstrate that PCB is the molecule responsible for C-phycocyanin's nephroprotective action in the acute kidney injury model caused by HgCl2. PCB was purified from C-phycocyanin and characterized by spectroscopy and mass spectrometry methods. Thirty-six male mice were administrated with 0.75, 1.5, or 3 mg per kg per d of PCB 30 min before the 5 mg kg-1 HgCl2 administration. PCB was administered during the following five days, after which the mice were euthanized. Kidneys were dissected to determine oxidative stress and redox environment markers, first-line antioxidant enzymes, effector caspase activities, and kidney damage markers.The quality of purified PCB was evaluated by spectroscopy and mass spectrometry. All PCB doses prevented alterations in oxidative stress markers, antioxidant enzymes, and caspase 9 activities. However, only the dose of 3 mg per kg per d PCB avoided the redox environment disturbance produced by mercury. All doses of PCB partially prevented the down-expression of nephrin and podocin with a consequent reduction in the damage score in a dose-effect manner. In conclusion, it was proven that phycocyanobilin is the molecule responsible for C-phycocyanin's nephroprotective action on acute kidney injury caused by mercury.

Antioxidant activity of proteins extracted from red alga dulse harvested in Japan.

In this study, we investigated antioxidant activity of proteins from the red alga dulse (Palmaria sp.) harvested in Hokkaido, Japan. The dulse proteins that contain phycoerythrin (PE) as the main component showed a high radical scavenging activity. To clarify the key constituent of antioxidant activity in dulse proteins, we prepared recombinant dulse PE ß-subunit (rPEß) (apoprotein) and chromophores from the dulse proteins. As a result, the rPEß showed lower radical scavenging activity than that of dulse proteins. On the other hand, the dulse chromophores composed mainly of phycoerythrobilin (PEB) indicated extremely higher radical scavenging activity (90.4% ± 0.1%) than that of dulse proteins (17.9% ± 0.1%) on ABTS assay. In addition, on cell viability assay using human neuroblastoma SH-SY5Y cells, the dulse chromophores showed extracellular and intracellular cytoprotective effects against H2 O2 -induced cell damage. From these data, we concluded that the dulse proteins have antioxidant ability and the activity principally derives from the chromophores. PRACTICAL APPLICATION: Dulse is an abundant and underused resource, which contains a lot of proteins, especially phycoerythrin. We here demonstrated that the practically prepared dulse proteins possessed antioxidant activity and clarified that chromophores from the dulse proteins were the key components. Therefore, the dulse proteins have a potential for functional material.

Co-administration of Phycocyanobilin and/or Phase 2-Inducer Nutraceuticals for Prevention of Opiate Tolerance.

Chronic use of opiates for control of chronic pain is complicated by the development of tolerance and hyperalgesia, and hence usually entails dose escalation and diminished efficacy. Our evolving understanding of the mechanisms mediating induction of morphine tolerance may enable discovery of adjunct measures which can prevent this tolerance; this essay proposes that certain nutraceuticals may have utility in this regard. Considerable evidence now points to an obligate role for production of peroxynitrite and other oxidants in the dorsal horn in development of morphine tolerance. Various isoforms of NADPH oxidase are the chief source of the superoxide which gives rise to these oxidants. Since heme oxygenase, via its products bilirubin and carbon monoxide, functions as a physiological inhibitor of various isoforms of NADPH oxidase, phase 2-inducing nutraceuticals with blood brain-barrier permeability such as lipoic acid, an effective inducer of heme oxygenase-1, may have potential for prevention of morphine tolerance; indeed, this has been demonstrated in a mouse study. The phycocyanobilin (PhyCB) chromophore of spirulina, a structural analog of biliverdin, shares bilirubin's ability to inhibit NAPDH oxidase complexes; hence, administration of spirulina or of PhyCB-enriched spirulina extracts merits evaluation in rodent models of morphine tolerance. Uric acid quenches peroxynitrite-derived radicals, and its plasma level can be boosting via supplementation with inosine; indeed, administration of inosine has been shown to counteract development of hyperalgesia in rodents. If practical doses of these agents can be shown to prevent morphine tolerance and hyperalgesia in rodents, their use as adjuvants to clinical opiate therapy should be assessed.

Stabilization of Human Serum Albumin by the Binding of Phycocyanobilin, a Bioactive Chromophore of Blue-Green Alga Spirulina: Molecular Dynamics and Experimental Study.

Phycocyanobilin (PCB) binds with high affinity (2.2 x 106 M-1 at 25°C) to human serum albumin (HSA) at sites located in IB and IIA subdomains. The aim of this study was to examine effects of PCB binding on protein conformation and stability. Using 300 ns molecular dynamics (MD) simulations, UV-VIS spectrophotometry, CD, FT-IR, spectrofluorimetry, thermal denaturation and susceptibility to trypsin digestion, we studied the effects of PCB binding on the stability and rigidity of HSA, as well as the conformational changes in PCB itself upon binding to the protein. MD simulation results demonstrated that HSA with PCB bound at any of the two sites showed greater rigidity and lower overall and individual domain flexibility compared to free HSA. Experimental data demonstrated an increase in the α-helical content of the protein and thermal and proteolytic stability upon ligand binding. PCB bound to HSA undergoes a conformational change to a more elongated conformation in the binding pockets of HSA. PCB binding to HSA stabilizes the structure of this flexible transport protein, making it more thermostable and resistant to proteolysis. The results from this work explain at molecular level, conformational changes and stabilization of HSA structure upon ligand binding. The resultant increased thermal and proteolytic stability of HSA may provide greater longevity to HSA in plasma.

Suppression of human alloreactive T cells by linear tetrapyrroles; relevance for transplantation.

The main limitation to successful transplantation is the antigraft response developed by the recipient immune system, and the adverse side effects of immunosuppressive agents which are associated with significant toxicity and counter indications such as infection and cancer. Furthermore, immunosuppressants do little to prevent ischemia-reperfusion injury during the transplantation procedure itself hence there is a growing need to develop novel immunosuppressive drugs specifically aimed at prolonging graft survival. Linear tetrapyrroles derived from the breakdown of mammalian heme have been shown in numerous studies to play a protective role in allograft transplantation and ischemia-reperfusion injury; however, commercial sources of these products have not been approved for use in humans. Plants and algae produce equivalent linear tetrapyrroles called bilins that serve as chromophores in light-sensing. One such marine-derived tetrapyrrole, phycocyanobilin (PCB), shows significant structural similarity to mammalian biliverdin (BV) and may prove to be a safer alternative for use in the clinic if it can exert direct effects on human immune cells. Using a mixed lymphocyte reaction, we quantified the allogeneic responses of recipient cells to donor cells and found that PCB, like BV, effectively suppressed proliferation and proinflammatory cytokine production. In addition, we found that BV and PCB can directly downregulate the proinflammatory responses of both innate dendritic cells and adaptive T cells. We therefore propose that PCB may be an effective therapeutic drug in the clinical setting of transplantation and may also have wider applications in regulating inappropriate inflammation.

Phycocyanobilin accelerates liver regeneration and reduces mortality rate in carbon tetrachloride-induced liver injury mice.

AIM: To investigate the hepatoprotective effects of phycocyanobilin (PCB) in reducing hepatic injury and accelerating hepatocyte proliferation following carbon tetrachloride (CCl4) treatment. METHODS: C57BL/6 mice were orally administered PCB 100 mg/kg for 4 d after CCl4 injection, and then the serum and liver tissue of the mice were collected at days 1, 2, 3, 5 and 7 after CCl4 treatment. A series of evaluations were performed to identify the curative effects on liver injury and recovery. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin and superoxide dismutase (SOD) were detected to indirectly assess the anti-inflammatory effects of PCB. Meanwhile, we detected the expressions of hepatocyte growth factor, transforming growth factor alpha (TGF-α), TGF-ß, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), the factors which are associated with inflammation and liver regeneration. The protein expressions of proliferating cell nuclear antigen (PCNA), TNF-α and cytochrome C were detected by western blot. Furthermore, the survival rates were analyzed of mice which were administered a lethal dose of CCl4 (2.6 mg/kg) with or without PCB. RESULTS: In our research, PCB showed a strongly anti-inflammatory effect on CCl4-induced liver injury in mice. The ALT was significantly decreased after CCl4 treatment from day 1 (P < 0.01) and the AST was significantly decreased from day 2 (P < 0.001). Both albumin and liver SOD were increased from day 2 (P < 0.001 and P < 0.01), but serum SOD levels did not show a significant increase (P > 0.05). PCB protected the structure of liver from the injury by CCl4. TUNEL assay showed that PCB dramatically reduced the number of apoptotic cells after CCl4 treatment compared to the control (101.0 ± 25.4 vs 25.7 ± 6.4, P < 0.01). The result of western blotting showed that PCB could increase PCNA expression, decrease TNF-α and cytochrome C expression. Furthermore, data shows that PCB could improve the survival rate of acute liver failure (ALF) mice which were injected with a lethal dose of CCl4 (60.0% vs 20.0%). CONCLUSION: Our study indicated that PCB could be an ideal candidate for reversing acute liver injury or ALF.

Cell cycle regulation and apoptotic cell death in experimental colon carcinogenesis: intervening with cyclooxygenase-2 inhibitors.

Relative imbalance in the pathways regulating cell cycle, cell proliferation, or cell death marks a prerequisite for neoplasm. C-phycocyanin, a biliprotein from Spirulina platensis and a selective COX-2 inhibitor along with piroxicam, a traditional nonsteroidal antiinflammatory drug was used to investigate the role of cell cycle regulatory proteins and proinflammatory transcription factor NFκB in 1,2-dimethylhydrazine dihydrochloride (DMH)-induced rat colon carcinogenesis. Cell cycle regulators [cyclin D1, cyclin E, cyclin dependent kinase 2 (CDK2), CDK4, and p53], NFκB (p65) pathway, and proliferating cell nuclear antigen (PCNA) were evaluated by gene and protein expression, whereas apoptosis was studied by terminal deoxynucleotidyl transferase dUTP nick end labeling and apoptotic bleb assay. Molecular docking of ligand protein interaction was done to validate the in vivo results. Cyclin D1, cyclin E, CDK2, and CDK4 were overexpressed in DMH, whereas piroxicam and c-phycocyanin promoted the cell cycle arrest by downregulating them. Both drugs mediated apoptosis through p53 activation. Piroxicam and c-phycocyanin also stimulated antiproliferation by restraining PCNA expression and reduced cell survival via inhibiting NFκB (p65) pathway. Molecular docking revealed that phycocyanobilin (a chromophore of c-phycocyanin) interact with DNA binding site of NFκB. Inhibition of cyclin/CDK complex by piroxicam and c-phycocyanin affects the expression of p53 in colon cancer followed by downregulation of NFκB and PCNA levels, thus substantiating the antineoplastic role of these agents.

Carvedilol and spirulina may provide important health protection to smokers and other nicotine addicts: a call for pertinent research.

Nicotine and soluble semi-stable aldehydes and ketones in cigarette smoke are key mediators of the elevated risks for vascular disease, cancer, and chronic obstructive pulmonary disease observed in smokers. Nicotine, via sympathetic stimulation, increases risk for both vascular disease and cancer. Comprehensive suppression of sympathetic activity with the well-tolerated drug carvedilol, which inhibits betal 1, beta2 and alphal adrenergic receptors, may be protective to smokers and other nicotine addicts. The soluble aldehydes and ketones in tobacco smoke appear to exert their adverse effects through activation of NADPH oxidase complexes in vascular tissues and in the lungs. The phytochemical phycocyanobilin (PhyCB), richly supplied by the edible cyanobacterium spirulina, in studies on rodents and in human cell cultures has shown the ability to safely mimic intracellular bilirubin's physiological role as an inhibitor ofNADPH oxidase activity. It therefore may have potential for mitigating the pro-oxidative effects of tobacco smoke aldehydes and ketones. Joint administration of carvedilol and spirulina merits exploration as a strategy for moderating the pathogenic impact of smoking in chronic tobacco users who either fail to quit or refuse to try cessation of tobacco. Carvedilol may be appropriate for those who manage a nicotine addiction in other ways (smokeless tobacco, e-cigarettes, nicotine gum). Further clinical studies to evaluate the impact of carvedilol on cardiovascular risk factors in nicotine addicts, and rodent studies to assess markers of lung inflammation in smoke- exposed rodents fed PhyCB, are recommended.

Phycocyanobilin promotes PC12 cell survival and modulates immune and inflammatory genes and oxidative stress markers in acute cerebral hypoperfusion in rats.

Since the inflammatory response and oxidative stress are involved in the stroke cascade, we evaluated here the effects of Phycocyanobilin (PCB, the C-Phycocyanin linked tetrapyrrole) on PC12 cell survival, the gene expression and the oxidative status of hypoperfused rat brain. After the permanent bilateral common carotid arteries occlusion (BCCAo), the animals were treated with saline or PCB, taking samples 24h post-surgery. Global gene expression was analyzed with GeneChip Rat Gene ST 1.1 from Affymetrix; the expression of particular genes was assessed by the Fast SYBR Green RT-PCR Master Mix and Bioplex methods; and redox markers (MDA, PP, CAT, SOD) were evaluated spectrophotometrically. The PCB treatment prevented the H2O2 and glutamate induced PC12 cell injury assessed by the MTT assay, and modulated 190 genes (93 up- and 97 down-regulated) associated to several immunological and inflammatory processes in BCCAo rats. Furthermore, PCB positively modulated 19 genes mostly related to a detrimental pro-inflammatory environment and counteracted the oxidative imbalance in the treated BCCAo animals. Our results support the view of an effective influence of PCB on major inflammatory mediators in acute cerebral hypoperfusion. These results suggest that PCB has a potential to be a treatment for ischemic stroke for which further studies are needed.

Phycocyanin and phycocyanobilin from Spirulina platensis protect against diabetic nephropathy by inhibiting oxidative stress.

We and other investigators have reported that bilirubin and its precursor biliverdin may have beneficial effects on diabetic vascular complications, including nephropathy, via its antioxidant effects. Here, we investigated whether phycocyanin derived from Spirulina platensis, a blue-green algae, and its chromophore phycocyanobilin, which has a chemical structure similar to that of biliverdin, protect against oxidative stress and renal dysfunction in db/db mice, a rodent model for Type 2 diabetes. Oral administration of phycocyanin (300 mg/kg) for 10 wk protected against albuminuria and renal mesangial expansion in db/db mice, and normalized tumor growth factor-ß and fibronectin expression. Phycocyanin also normalized urinary and renal oxidative stress markers and the expression of NAD(P)H oxidase components. Similar antioxidant effects were observed following oral administration of phycocyanobilin (15 mg/kg) for 2 wk. Phycocyanobilin, bilirubin, and biliverdin also inhibited NADPH dependent superoxide production in cultured renal mesangial cells. In conclusion, oral administration of phycocyanin and phycocyanobilin may offer a novel and feasible therapeutic approach for preventing diabetic nephropathy.

Light-mediated control of DNA transcription in yeast.

A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light/cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems.

Assembly of synthetic locked phycocyanobilin derivatives with phytochrome in vitro and in vivo in Ceratodon purpureus and Arabidopsis.

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red light-absorbing form, Pr, and the far-red-light-absorbing form, Pfr. Here we performed in vitro and in vivo studies using locked phycocyanobilin derivatives, termed 15 Z anti phycocyanobilin (15ZaPCB) and 15 E anti PCB (15EaPCB). Recombinant bacterial and plant phytochromes incorporated either chromophore in a noncovalent or covalent manner. All adducts were photoinactive. The absorption spectra of the 15ZaPCB and 15EaPCB adducts were comparable with those of the Pr and Pfr form, respectively. Feeding of 15EaPCB, but not 15ZaPCB, to protonemal filaments of the moss Ceratodon purpureus resulted in increased chlorophyll accumulation, modulation of gravitropism, and induction of side branches in darkness. The effect of locked chromophores on phytochrome responses, such as induction of seed germination, inhibition of hypocotyl elongation, induction of cotyledon opening, randomization of gravitropism, and gene regulation, were investigated in wild-type Arabidopsis thaliana and the phytochrome-chromophore-deficient long hypocotyl mutant hy1. All phytochrome responses were induced in darkness by 15EaPCB, not only in the mutant but also in the wild type. These studies show that the 15Ea stereochemistry of the chromophore results in the formation of active Pfr-like phytochrome in the cell. Locked chromophores might be used to investigate phytochrome responses in many other organisms without the need to isolate mutants. The induction of phytochrome responses in the hy1 mutant by 15EaPCB were however less efficient than by red light irradiation given to biliverdin-rescued seeds or seedlings.

Taste-mediated behavioral and electrophysiological responses by the predatory fish Ariopsis felis to deterrent pigments from Aplysia californica ink.

Chemical defenses are used by many organisms to avoid predation, and these defenses may function by stimulating predators' chemosensory systems. Our study examined detection mechanisms for components of defensive ink of sea hares, Aplysia californica, by predatory sea catfish, Ariopsis felis. Behavioral analyses show aplysioviolin and phycoerythrobilin are detected intra-orally and by barbels and are deterrent at concentrations as low as 0.1% full strength. We performed electrophysiological recordings from the facial-trigeminal nerve complex innervating the maxillary barbel and tested aplysioviolin, phycoerythrobilin, amino acids, and bile salts in cross-adaptation experiments. Amino acids and bile salts are known stimulatory compounds for teleost taste systems. Our results show aplysioviolin and phycoerythrobilin are equally stimulatory and completely cross-adapt to each other's responses. Adaptation to aplysioviolin or phycoerythrobilin reduced but did not eliminate responses to amino acids or bile salts. Adaptation to amino acids or bile salts incompletely reduced responses to aplysioviolin or phycoerythrobilin. The fact that cross-adaptations with aplysioviolin and phycoerythrobilin were not completely reciprocal indicates there are amino acid and bile salt sensitive fibers insensitive to aplysioviolin and phycoerythrobilin. These results indicate two gustatory pathways for aplysioviolin and phycoerythrobilin: one independent of amino acids and bile salts and another shared with some amino acids.

Clinical potential of phycocyanobilin for induction of T regulatory cells in the management of inflammatory disorders.

Exposure of human mononuclear cells to phycocyanin in vitro is reported to promote generation of Treg cells. Induction of heme oxygenase-1 (HO-1) in lymphocytes has a similar effect, and it is not likely to be accidental that a key product of HO-1 activity, biliverdin, is homologous to the structure of phycocyanin's chromophore phycocyanobilin (PhyCB). Moreover, Treg induction is observed in mice injected with bilirubin, biliverdin's chief metabolite. These considerations suggest that bilirubin, generated within lymphocytes by HO-1 activation, may play a physiological role in the promotion of Treg immunomodulation. This effect of bilirubin is likely to be independent of NADPH oxidase inhibition, since the NAPDH oxidase activity of macrophages is necessary for Treg induction, possibly because it contributes to HO-1 induction in lymphocytes. In light of numerous reports that oral phycocyanin is beneficial in various rodent models of autoimmune disorders, it is reasonable to suspect that PhyCB-enriched spirulina extracts may have clinical potential for boosting Treg activity in human autoimmune or allergic syndromes, mimicking the physiological role of HO-1 induction in this regard.