Phytochromes are widely occurring red/far-red photoreceptors that utilize a linear tetrapyrrole (bilin) chromophore covalently bound within a knotted PAS-GAF domain pair. Cyanobacteria also contain more distant relatives of phytochromes that lack this knot, such as the phytochrome-related cyanobacteriochromes implicated to function as blue/green switchable photoreceptors. In this study, we characterize the cyanobacteriochrome Tlr0924 from the thermophilic cyanobacterium Thermosynechococcus elongatus. Full-length Tlr0924 exhibits blue/green photoconversion across a broad range of temperatures, including physiologically relevant temperatures for this organism. Spectroscopic characterization of Tlr0924 demonstrates that its green-absorbing state is in equilibrium with a labile, spectrally distinct blue-absorbing species. The photochemically generated blue-absorbing state is in equilibrium with another species absorbing at longer wavelengths, giving a total of 4 states. Cys499 is essential for this behavior, because mutagenesis of this residue results in red-absorbing mutant biliproteins. Characterization of the C 499 D mutant protein by absorbance and CD spectroscopy supports the conclusion that its bilin chromophore adopts a similar conformation to the red-light-absorbing P r form of phytochrome. We propose a model photocycle in which Z/E photoisomerization of the 15/16 bond modulates formation of a reversible thioether linkage between Cys499 and C10 of the chromophore, providing the basis for the blue/green switching of cyanobacteriochromes.Photosynthetic organisms face the need to coordinate their metabolic responses to their light environment, so that photosynthesis and redox balance are properly maintained for growth. This is accomplished by a wide range of photosensory proteins (1,2). The first such proteins to be discovered were the phytochromes, which are red/far-red photosensors initially described in plants and later shown to be widespread in both photosynthetic and nonphotosynthetic organisms (3,4). Upon excitation with red light, phytochromes photoconvert from the redabsorbing P r state 1 , which is usually thermally stable, to the far-red absorbing P fr state (5). This reversible interconversion is the result of light-driven Z/E isomerization of the 15/16 double bond of the protein-bound bilin chromophore ( Fig. 1), which is covalently attached to a Cys residue in the conserved photosensory core of phytochromes. This photosensory core is generally found N-terminal to putative output domains implicated in signal transduction, such as the histidine kinase domain of the cyanobacterial phytochrome Cph1 (6). Since both P r and NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 July 8. Published in final edited form as:Biochemistry. 2008 July 8; 47(27): 7304-7316. doi:10.1021/bi800088t. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript P fr states can generate signaling outputs (4,7), light modulates the signaling activity of phytochrome...

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The role of nitric oxide (NO) and free radicals in the development of microvascular disease in type 1 diabetes remains unclear. We have measured NO and isoprostane (a stable marker of in vivo lipid peroxidation) production in 13 type 1 diabetic subjects with normal urinary albumin excretion and 13 healthy volunteers. Whole-body NO synthesis was quantified by measuring the urinary excretion of 15 N-nitrate after the intravenous administration of L-[15 N] 2 -arginine. The urinary excretion of the major urinary metabolite of 15-F 2t -isoprostane (8-iso-prostaglandin-F 2␣ ), 2,3-dinor-5,6-dihydro-F 2t -IsoP, was quantified as a marker of in vivo lipid peroxidation. Whole-body NO synthesis was significantly higher in diabetic subjects compared with control subjects (342 vs. 216 nmol 15 N-nitrate/mmol creatinine [95% CI of the difference 45-207], P = 0.005). This increase was not explained by a difference in renal function between the 2 groups. There was no difference in 2,3-dinor-5,6-dihydro-F 2t -IsoP excretion between diabetic subjects and control subjects (44.8 ± 7.8 vs. 41.4 ± 10.0 ng/mmol creatinine, mean ± 95% CI). However, there was an inverse correlation between NO synthesis and free radical activity in subjects with diabetes (r = -0.62, P = 0.012) that was not observed in control subjects (r = 0.37, P = 0.107). We conclude that whole-body NO synthesis is higher in type 1 diabetic subjects with normal urinary albumin excretion than in control subjects. The inverse correlation between isoprostane production and NO synthesis in diabetic subjects is consistent with the hypothesis that NO is being inactivated by reactive oxygen species. Diabetes 49:857-862, 2000 T he Diabetes Control and Complications Trial has confirmed that tight blood glucose control significantly delays the onset and slows the progression of microvascular complications in type 1 diabetes (1). However, despite optimum blood glucose control, a substantial proportion of diabetic subjects in this study developed serious microvascular complications. In contrast, in the Wisconsin Epidemiological Study, a significant proportion (30%) of subjects with type 1 diabetes did not manifest severe microvascular complications after 10 years of follow-up, although they had chronic poorly controlled diabetes (2). It would be useful if we could understand this discrepancy underlying the development of microvascular disease so that those at risk could be identified. The benefit/risk ratio of maintaining tight blood glucose control could be increased if targeted toward individuals at greatest risk of complications due to hyperglycemia.The complex issues involved in both the pathogenesis of and individual susceptibility to the angiopathy associated with type 1 diabetes have been well documented (3-5). The endothelium and, in particular, the production of nitric oxide (NO) have been the subject of considerable research in recent years. NO maintains basal vasodilator tone, is a potent platelet anti-aggregant, reduces the ability of monocytes to adhere to endothelial...

Case Presentation A 64-year-old male with a 15-year history of poorly controlled type 2 diabetes and a 10-year history of hypertension and hyperlipidemia had developed multiple diabetes-related complications within the last 5 years. He first developed albuminuria 5 years ago, and over the next several years experienced fairly rapid decline in kidney function, with eGFR of 55 mL/min/1.73m2 noted 2 years ago. He was diagnosed with proliferative retinopathy 5 years ago and underwent laser photocoagulation. Four years ago, he noted symptoms of peripheral neuropathy manifested as shooting pain and numbness with loss of light touch, thermal and vibratory sensation in a stocking distribution. Last year he developed a non-healing ulcer on the plantar aspect of his left foot which was complicated with gangrene and resulted in a below-the-knee amputation of the left leg one year ago. He now reports a new onset of weakness, lightheadedness and dizziness on standing that affects his daily activities. He reports lancinating pain in his right lower extremity, worse in the evening. Medications include: neutral protamine Hagedorn insulin twice daily and regular insulin on a sliding scale, metoprolol 50 mg/d, lisinopril 40 mg/d, atorvastatin 80 mg/d, furosemide 40 mg/d and aspirin 81 mg/d. Blood pressure is 127/69 mm Hg with a pulse rate of 96 bpm while supine and 94/50 mmHg with a pulse rate of 102 bpm while standing. Strength is normal but with a complete loss of all sensory modalities to the knee in his remaining limb and up to the wrists in both upper extremities, and he is areflexic. Today's laboratory evaluations show a serum creatinine of 2.8 mg/dl, an estimated GFR (eGFR) of 24 ml/min/1.73m2, a hemoglobin A1c (HbA1c) of 7.9 % and 2.1 g of urine protein per gram of creatinine. What would be the most appropriate management for this patient?