Engineering tyrosine-based electron flow pathways in proteins: The case of aplysia myoglobin

Tyrosine residues can act as redox cofactors that provide an electron transfer ("hole-hopping") route that enhances the rate of ferryl heme iron reduction by externally added reductants, for example, ascorbate. Aplysia fasciata myoglobin, having no naturally occurring tyrosines but 15 phenylalanines that can be selectively mutated to tyrosine residues, provides an ideal protein with which to study such through-protein electron transfer pathways and ways to manipulate them. Two surface exposed phenylalanines that are close to the heme have been mutated to tyrosines (F42Y, F98Y). In both of these, the rate of ferryl heme reduction increased by up to 3 orders of magnitude. This result cannot be explained in terms of distance or redox potential change between donor and acceptor but indicates that tyrosines, by virtue of their ability to form radicals, act as redox cofactors in a new pathway. The mechanism is discussed in terms of the Marcus theory and the specific protonation/deprotonation states of the oxoferryl iron and tyrosine. Tyrosine radicals have been observed and quantified by EPR spectroscopy in both mutants, consistent with the proposed mechanism. The location of each radical is unambiguous and allows us to validate theoretical methods that assign radical location on the basis of EPR hyperfine structure. Mutation to tyrosine decreases the lipid peroxidase activity of this myoglobin in the presence of low concentrations of reductant, and the possibility of decreasing the intrinsic toxicity of hemoglobin by introduction of these pathways is discussed. © 2012 American Chemical Society

Highly efficient separation of actinides from lanthanides by a phenanthroline-derived bis-triazine ligand

The synthesis, lanthanide complexation, and solvent ex- traction of actinide(III) and lanthanide(III) radiotracers from nitric acid solutions by a phenanthroline-derived quadridentate bis-triazine ligand are described. The ligand separates Am(III) and Cm(III) from the lanthanides with remarkably high efficiency, high selectivity, and fast extraction kinetics compared to its 2,2'-bipyridine counterpart. Structures of the 1:2 bis-complexes of the ligand with Eu(III) and Yb(III) were elucidated by X-ray crystallography and force field calculations, respec-tively. The Eu(III) bis-complex is the first 1:2 bis-complex of a quadridentate bis-triazine ligand to be characterized by crystallography. The faster rates of extraction were verified by kinetics measurements using the rotating membrane cell technique in several diluents. The improved kinetics of metal ion extraction are related to the higher surface activity of the ligand at the phase interface. The improvement in the ligand's properties on replacing the bipyridine unit with a phenanthroline unit far exceeds what was anticipated based on ligand design alone

C-Peptide Increases Na,K-ATPase Expression via PKC- and MAP Kinase-Dependent Activation of Transcription Factor ZEB in Human Renal Tubular Cells

Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC) in control and hyperglycemic conditions.HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86)Rb(+)) uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM). DNA binding activity was determined by electrical mobility shift assay (EMSA). Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α(1)-subunit protein expression, accompanied with increase in (86)Rb(+) uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1)-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6), concomitant with Na,K-ATPase α(1)-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(1)-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing.Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription factor that has not been previously implicated in C-peptide-mediated signaling. Importantly, only physiological concentrations of C-peptide elicit this effect

Proinsulin C-peptide elicits disaggregation of insulin resulting in enhanced physiological insulin effects

Using surface plasmon resonance (SPR) and electrospray mass spectrometry (ESI-MS), proinsulin C-peptide was found to influence insulin-insulin interactions. In SPR with chip-bound insulin, C-peptide mixed with analyte insulin increased the binding, while alone C-peptide did not. A control peptide with the same residues in random sequence had little effect. In ESI-MS, C-peptide lowered the presence of insulin hexamer. The data suggest that C-peptide promotes insulin disaggregation. Insulin/insulin oligomer μM dissociation constants were determined. Compatible with these findings, type 1 diabetic patients receiving insulin and C-peptide developed 66% more stimulation of glucose metabolism than when given insulin alone. A role of C-peptide in promoting insulin disaggregation may be important physiologically during exocytosis of pancreatic β-cell secretory granulae and pharmacologically at insulin injection sites. It is compatible with the normal co-release of C-peptide and insulin and may contribute to the beneficial effect of C-peptide and insulin replacement in type 1 diabetics

Transplantation of canine olfactory ensheathing cells producing chondroitinase ABC promotes chondroitin sulphate proteoglycan digestion and axonal sprouting following spinal cord injury

Olfactory ensheathing cell (OEC) transplantation is a promising strategy for treating spinal cord injury (SCI), as has been demonstrated in experimental SCI models and naturally occurring SCI in dogs. However, the presence of chondroitin sulphate proteoglycans within the extracellular matrix of the glial scar can inhibit efficient axonal repair and limit the therapeutic potential of OECs. Here we have used lentiviral vectors to genetically modify canine OECs to continuously deliver mammalian chondroitinase ABC at the lesion site in order to degrade the inhibitory chondroitin sulphate proteoglycans in a rodent model of spinal cord injury. We demonstrate that these chondroitinase producing canine OECs survived at 4 weeks following transplantation into the spinal cord lesion and effectively digested chondroitin sulphate proteoglycans at the site of injury. There was evidence of sprouting within the corticospinal tract rostral to the lesion and an increase in the number of corticospinal axons caudal to the lesion, suggestive of axonal regeneration. Our results indicate that delivery of the chondroitinase enzyme can be achieved with the genetically modified OECs to increase axon growth following SCI. The combination of these two promising approaches is a potential strategy for promoting neural regeneration following SCI in veterinary practice and human patients

Spectra of W39+^{39+}-W47+^{47+} in the 12 nm to 20 nm region observed with an EBIT light source

We observed spectra of highly ionized tungsten in the extreme ultraviolet with an electron beam ion trap (EBIT) and a grazing incidence spectrometer at the National Institute of Standards and Technology. Stages of ionization were distinguished by varying the energy of the electron beam between 2.1 keV and 4.3 keV and correlating the energies with spectral line emergence. The spectra were calibrated by reference lines of highly ionized iron produced in the EBIT. Identification of the observed lines was aided by collisional-radiative modeling of the EBIT plasma. Good quantitative agreement was obtained between the modeling results and the experimental observations. Our line identifications complement recent results for W$^{40+}$-W$^{45+}$ observed in a tokamak plasma by P\"{u}tterich {\it et al} (\jpb {\bf 38}, 3071, 2005). For most lines we agree with their assignment of ionization stage. Additionally, we present new identifications for some allowed and forbidden lines of W$^{39+}$, W$^{44+}$, W$^{46+}$, and W$^{47+}$. The uncertainties of our wavelengths range from 0.002 nm to 0.010 nm.Comment: 19 pages, 8 figure

Liver Graft Revascularization by Donor Portal Vein Arterialization Following “No Touch” Donor Hepatectomy

Unsatisfactory immediate function of the transplanted liver together with technical complications contribute to a persisting early mortality for hepatic transplantation in the 20% range. We report our initial clinical experience with methods, one not previously used clinically, that resulted in uniformly well-functioning liver grafts in 11 patients and contributed to a satisfactory success rate for the procedure. Donors were heart-beating. During the donor operation all manipulations of the liver were avoided until after cold preservation, achieved by external cooling at the same time as circulatory interruption, donor exsanguination and perfusion of the liver with cold oxygenated fluid of “extracellular̵ type. The organs were then gently dissected. At transplantation the livers were revascularized with arterial blood shunted from the recipient iliac artery to the graft portal vein after completion of the suprahepatic IVC anastomosis. The infrahepatic IVCs and hepatic arteries were then joined, the iliac artery shunts discontinued and the portal veins joined. Total ischaemic intervals for the allografts were 3½–8 (average 5). Anhepatic intervals were 1–2¼ (average 2). The arterio-portal shunts were operating for 18–85 (mean 46) min. Blood loss and haemodynamic, acid-base and electrolyte abnormalities at revascularization were minimal. All grafts secreted bile immediately and all parameters reflected continuing improvement of liver function thereafter. Nine patients (82%) are alive between 4 and 18 (mean 11) months after transplantation. We conclude that these methods offer effective avoidance of serious organ damage during donor hepatectomy and preservation, reduced allograft ischaemic interval and reduced recipient anhepatic time. They result in avoidance of blood loss at the time of revascularization, together with minimal haemodynamic, acid-base or biochemical changes. In addition, they allow the surgeon to perform and test all anastomoses without time constraints, provide the capability to deal with unexpected complications, and assure good early graft function

Effect of sirolimus on malignancy and survival after kidney transplantation: systematic review and meta-analysis of individual patient data

Objective To examine risk of malignancy and death in patients with kidney transplant who receive the immunosuppressive drug sirolimus.Design Systematic review and meta-analysis of individual patient data.Data sources Medline, Embase, and the Cochrane Central Register of Controlled Trials from inception to March 2013.Eligibility Randomized controlled trials comparing immunosuppressive regimens with and without sirolimus in recipients of kidney or combined pancreatic and renal transplant for which the author was willing to provide individual patient level data. Two reviewers independently screened titles/abstracts and full text reports of potentially eligible trials to identify studies for inclusion. All eligible trials reported data on malignancy or survival.Results the search yielded 2365 unique citations. Patient level data were available from 5876 patients from 21 randomized trials. Sirolimus was associated with a 40% reduction in the risk of malignancy (adjusted hazard ratio 0.60, 95% confidence interval 0.39 to 0.93) and a 56% reduction in the risk of non-melanoma skin cancer (0.44, 0.30 to 0.63) compared with controls. the most pronounced effect was seen in patients who converted to sirolimus from an established immunosuppressive regimen, resulting in a reduction in risk of malignancy (0.34, 0.28 to 0.41), non-melanoma skin cancer (0.32, 0.24 to 0.42), and other cancers (0.52, 0.38 to 0.69). Sirolimus was associated with an increased risk of death (1.43, 1.21 to 1.71) compared with controls.Conclusions Sirolimus was associated with a reduction in the risk of malignancy and non-melanoma skin cancer in transplant recipients. the benefit was most pronounced in patients who converted from an established immunosuppressive regimen to sirolimus. Given the risk of mortality, however, the use of this drug does not seem warranted for most patients with kidney transplant. Further research is needed to determine if different populations, such as those at high risk of cancer, might benefit from sirolimus.PfizerOttawa Hosp, Res Inst, Ottawa, ON K1H 7W9, CanadaUniv Ottawa, Ottawa, ON, CanadaCairo Univ, Cairo Kidney Ctr, Cairo, EgyptLimites Med Res, Vacallo, SwitzerlandUniv Manitoba, Dept Pediat & Childs Hlth, Winnipeg, MB, CanadaLund Univ, Dept Nephrol & Transplantat, Malmo, SwedenUniversidade Federal de São Paulo, Hosp Rim & Hipertensao, São Paulo, BrazilAddenbrookes Hosp, Dept Renal Med, Cambridge, EnglandNorthwestern Univ, Feinberg Sch Med, Chicago, IL 60611 USAMaastricht Univ, Med Ctr, Maastricht, NetherlandsSt Louis Hosp, Dept Nephrol, Paris, FranceHosp JW Goethe, Div Nephrol, Frankfurt, GermanyUniv Munich, Dept Surg, Munich, GermanyGoethe Univ Frankfurt, JW Goethe Clin, Clin Dermatol Venerol & Allergol, Frankfurt, GermanyInst Klin Expt Med, Dept Nephrol, Prague, Czech RepublicUniv Cambridge, Addenbrookes Hosp, Dept Surg, NIHR Cambridge Biomed Res Ctr, Cambridge CB2 2QQ, EnglandUniversidade Federal de São Paulo, Hosp Rim & Hipertensao, São Paulo, BrazilWeb of Scienc

Towards an optimal therapy strategy for myogenous TMD, physiotherapy compared with occlusal splint therapy in an RCT with therapy-and-patient-specific treatment durations

Appendix. Threshold of signs and symptoms. Post-hoc power analysis on measures of effectiveness. Two-way ANOVA statistical analysis for pain intensity. Rules for progressing and ending splint therapy. Rules for progressing and ending physiotherapy. A stepped-care model including two possible therapies. (PDF 86 kb