Polyethylene Glycol Camouflaged Earthworm Hemoglobin.

Nearly 21 million components of blood and whole blood and transfused annually in the United States, while on average only 13.6 million units of blood are donated. As the demand for Red Blood Cells (RBCs) continues to increase due to the aging population, this deficit will be more significant. Despite decades of research to develop hemoglobin (Hb) based oxygen (O2) carriers (HBOCs) as RBC substitutes, there are no products approved for clinical use. Lumbricus terrestris erythrocruorin (LtEc) is the large acellular O2 carrying protein complex found in the earthworm Lumbricus terrestris. LtEc is an extremely stable protein complex, resistant to autoxidation, and capable of transporting O2 to tissue when transfused into mammals. These characteristics render LtEc a promising candidate for the development of the next generation HBOCs. LtEc has a short half-life in circulation, limiting its application as a bridge over days, until blood became available. Conjugation with polyethylene glycol (PEG-LtEc) can extend LtEc circulation time. This study explores PEG-LtEc pharmacokinetics and pharmacodynamics. To study PEG-LtEc pharmacokinetics, hamsters instrumented with the dorsal window chamber were subjected to a 40% exchange transfusion with 10 g/dL PEG-LtEc or LtEc and followed for 48 hours. To study the vascular response of PEG-LtEc, hamsters instrumented with the dorsal window chamber received multiple infusions of 10 g/dL PEG-LtEc or LtEc solution to increase plasma LtEc concentration to 0.5, then 1.0, and 1.5 g/dL, while monitoring the animals' systemic and microcirculatory parameters. Results confirm that PEGylation of LtEc increases its circulation time, extending the half-life to 70 hours, 4 times longer than that of unPEGylated LtEc. However, PEGylation increased the rate of LtEc oxidation in vivo. Vascular analysis verified that PEG-LtEc showed the absence of microvascular vasoconstriction or systemic hypertension. The molecular size of PEG-LtEc did not change the colloid osmotic pressure or blood volume expansion capacity compared to LtEc, due to LtEc's already large molecular size. Taken together, these results further encourage the development of PEG-LtEc as an O2 carrying therapeutic

S-nitrosocaptopril nanoparticles as nitric oxide-liberating and transnitrosylating anti-infective technology

Nitric oxide (NO) is an essential agent of the innate immune system which exhibits multi-mechanistic antimicrobial activity. Previously, increased antimicrobial activity against E. coli with nitric oxide releasing nanoparticles (NO-np) was demonstrated when combined with glutathione, versus NO-np alone, due to formation of S-nitrosoglutathione (GSNO), a potent transnitrosylating agent. To capitalize on this finding, the thiol-containing ACE-inhibitor, captopril, was incorporated into the NO-np system to form SNO-CAP-nps, nanoparticles that can both release NO as well as form S-nitrosocaptopril. SNO-CAP-nps demonstrated sustained release of NO and GSNO production in the presence of GSH over time. Both E. coli and MRSA were highly susceptible to SNO-CAP-np in a dose-dependent fashion, with E. coli being highly sensitive, while demonstrating no toxicity when exposed to zebrafish embryos. Given the significance of E. coli in urosepsis, as well as the ability of nanotechnology to overcome biofilm formation, SNO-CAP-np may prove useful against catheter-associated urinary tract infection.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/nanomedicine-nanotechnology-biology-and-medicineKeywords: Antibacterial, Nitric oxide, Nanotechnology, Nitrosothiols, E. col

Nitric oxide generating/releasing materials

Harnessing the impressive therapeutic potential of nitric oxide (NO) remains an ongoing challenge. This paper describes several of the current strategies both with respect to the underlying chemistry and physics and to the applications where they have shown promise. Included in this overview are molecular systems such as NONOates that release NO through chemical reactions and delivery vehicles such as nanoparticles that can generate, store, transport and deliver NO and related bioactive forms of NO such as nitrosothiols. Although there has been much positive movement, it is clear that we are only at the early stages of knowing how to precisely produce, transport and deliver to targeted sites therapeutic levels of NO and related molecules

FTDP-17 tau mutations decrease the susceptibility of tau to calpain I digestion

AbstractFrontal temporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) is caused by splice site and missense mutations in the tau gene, and characterized by the accumulation of filamentous tau in cerebral neurons and glia. The missense mutations reduce the ability of tau to promote microtubule assembly and increase the ability of tau to form filaments. In this report we demonstrate that mutants V337M and R406W are less susceptible than mutant P301L or corresponding wild type tau to degradation by calpain I. The differences were at least in part due to changes in accessibility of a cleavage site located about 100 amino acids off the carboxy-terminus. The results suggest that the pathogenesis of some forms of FTDP-17 may involve tau accumulation due to decreased proteolytic degradation

Symmetric interspecies hybrids of mouse and human hemoglobin: molecular basis of their abnormal oxygen affinity

Interspecies hybrids of HbA and Hb from mouse C57BL/10 [ α2M α2H and α2H α2M (H=human, M=mouse)], representing 19 and 27 sequence differences αβ per dimers (as compared with human αβ dimer) have been generatedin vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O2 binding. The intrinsic O2 affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid α2M α2H has high O2 affinity (compared to either human or mouse Hb), while the interspecies hybrid α2H α2M exhibits a very low O2 affinity. These results suggest that the mouse β chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse α and β chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse β chain. DPG binds the tetramer in the central cavity formed by the two β subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the α chain present in the tetramer. The mouse α chain reduces considerably the DPG right shift of the O2 affinity of the human β-chain containing hybrid. Sequence analysis suggest that perturbations of the α1 β1 (not the α1 β1) are communicated to the DPG binding pocket in the presence of the alien subunit, and are the primary determinant of the ligand binding properties. The results have implications for the design of Hb-based blood substitutes and understanding of the inhibitory potential of mouse α chains in transgenic mouse expressing human αS chains

Inhibition of sickle &#946;-Chain (&#946;<SUP>S</SUP>)-dependent polymerization by nonhuman &#945;-chains

Horse &#945;-chain inhibits sickle &#946;-chain-dependent polymerization; however, its inhibitory potential is not as high as that of mouse &#945;-chain. Horse &#945;-(1-30) and &#945;-(31-141) segments make, respectively, minor and major contributions to the inhibitory potential of horse &#945;-chain. The sum of the inhibitory potential of the two segments does not account for the inhibitory potential of the full-length horse &#945;-chain. Although the polymerization inhibitory potential of horse &#945;-chain is lower than mouse &#945;-chain, the inhibitory potential of horse &#945;-(31-141) is comparable to that of mouse &#945;-(31-141). When mouse &#945;-(1-30) is stitched to horse &#945;-(31-141), the product is a chimeric &#945;-chain with an inhibitory potential greater than mouse &#945;-chain. In contrast, the stitching of horse &#945;-(1-30) with mouse &#945;-(31-141) had no additional inhibitory potential. Molecular modeling studies of HbS containing the mouse-horse chimeric &#945;-chain indicate altered side-chain interactions at the &#945;1&#946;1 interface when compared with HbS. In addition, the AB/GH corner perturbations facilitate a different stereochemistry for the interaction of the &#949;-amino group of Lys-16(&#945;) with the &#946;-carboxyl group of Asp-116(&#945;), resulting in a decrease in the accessibility of the side chain of Lys-16(&#945;) to the solvent. Based on molecular modeling, we speculate that these perturbations by themselves, or in synergy with the altered conformational aspects of the &#945;1&#946;1interactions, represent the molecular basis of the superinhibitory potential of the mouse-horse chimeric &#945;-chains

Polyethylene Glycol Camouflaged Earthworm Hemoglobin.

Nearly 21 million components of blood and whole blood and transfused annually in the United States, while on average only 13.6 million units of blood are donated. As the demand for Red Blood Cells (RBCs) continues to increase due to the aging population, this deficit will be more significant. Despite decades of research to develop hemoglobin (Hb) based oxygen (O2) carriers (HBOCs) as RBC substitutes, there are no products approved for clinical use. Lumbricus terrestris erythrocruorin (LtEc) is the large acellular O2 carrying protein complex found in the earthworm Lumbricus terrestris. LtEc is an extremely stable protein complex, resistant to autoxidation, and capable of transporting O2 to tissue when transfused into mammals. These characteristics render LtEc a promising candidate for the development of the next generation HBOCs. LtEc has a short half-life in circulation, limiting its application as a bridge over days, until blood became available. Conjugation with polyethylene glycol (PEG-LtEc) can extend LtEc circulation time. This study explores PEG-LtEc pharmacokinetics and pharmacodynamics. To study PEG-LtEc pharmacokinetics, hamsters instrumented with the dorsal window chamber were subjected to a 40% exchange transfusion with 10 g/dL PEG-LtEc or LtEc and followed for 48 hours. To study the vascular response of PEG-LtEc, hamsters instrumented with the dorsal window chamber received multiple infusions of 10 g/dL PEG-LtEc or LtEc solution to increase plasma LtEc concentration to 0.5, then 1.0, and 1.5 g/dL, while monitoring the animals' systemic and microcirculatory parameters. Results confirm that PEGylation of LtEc increases its circulation time, extending the half-life to 70 hours, 4 times longer than that of unPEGylated LtEc. However, PEGylation increased the rate of LtEc oxidation in vivo. Vascular analysis verified that PEG-LtEc showed the absence of microvascular vasoconstriction or systemic hypertension. The molecular size of PEG-LtEc did not change the colloid osmotic pressure or blood volume expansion capacity compared to LtEc, due to LtEc's already large molecular size. Taken together, these results further encourage the development of PEG-LtEc as an O2 carrying therapeutic

Polyethylene Glycol Camouflaged Earthworm Hemoglobin.

Nearly 21 million components of blood and whole blood and transfused annually in the United States, while on average only 13.6 million units of blood are donated. As the demand for Red Blood Cells (RBCs) continues to increase due to the aging population, this deficit will be more significant. Despite decades of research to develop hemoglobin (Hb) based oxygen (O2) carriers (HBOCs) as RBC substitutes, there are no products approved for clinical use. Lumbricus terrestris erythrocruorin (LtEc) is the large acellular O2 carrying protein complex found in the earthworm Lumbricus terrestris. LtEc is an extremely stable protein complex, resistant to autoxidation, and capable of transporting O2 to tissue when transfused into mammals. These characteristics render LtEc a promising candidate for the development of the next generation HBOCs. LtEc has a short half-life in circulation, limiting its application as a bridge over days, until blood became available. Conjugation with polyethylene glycol (PEG-LtEc) can extend LtEc circulation time. This study explores PEG-LtEc pharmacokinetics and pharmacodynamics. To study PEG-LtEc pharmacokinetics, hamsters instrumented with the dorsal window chamber were subjected to a 40% exchange transfusion with 10 g/dL PEG-LtEc or LtEc and followed for 48 hours. To study the vascular response of PEG-LtEc, hamsters instrumented with the dorsal window chamber received multiple infusions of 10 g/dL PEG-LtEc or LtEc solution to increase plasma LtEc concentration to 0.5, then 1.0, and 1.5 g/dL, while monitoring the animals' systemic and microcirculatory parameters. Results confirm that PEGylation of LtEc increases its circulation time, extending the half-life to 70 hours, 4 times longer than that of unPEGylated LtEc. However, PEGylation increased the rate of LtEc oxidation in vivo. Vascular analysis verified that PEG-LtEc showed the absence of microvascular vasoconstriction or systemic hypertension. The molecular size of PEG-LtEc did not change the colloid osmotic pressure or blood volume expansion capacity compared to LtEc, due to LtEc's already large molecular size. Taken together, these results further encourage the development of PEG-LtEc as an O2 carrying therapeutic