Carbonated water mediated preparation of poly(N-isopropylacrylamide) thermoresponsive gels and liquids

Carbonic acid solution was used a medium for the free radical polymerization synthesis of poly( N-isopropylacrylamide) (p-nipam) thermoresponsive polymer as an alternative to conventional inert gas purging. It was found that p-nipam cross-linked gels or linear liquids and p-nipam/dextran magnetite composite gels could be very rapidly prepared and large gels recovered intact from open air vessels. A porogen was necessary for high thermoresponse, and dextran use resulted in microporous composite gels that gave optimal thermal response at weight ratio of p-nipam:dextran of 4:1. Up to 82% weight loss was rapidly obtained upon warming above the lower critical solution temperature. Analysis of products was made by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared (FT-IR), and superconducting quantum interference device (SQUID). A simplified and efficient overall method for preparation of biomedical polymers is shown. A wider application of H2CO3 solution as a viable alternative media and to allow open air aqueous polymerizations of water soluble or hydrophilic monomers is indicated.     

Immobilized alpha-melanocyte stimulating hormone 10-13 (GKPV) inhibits tumor necrosis factor-alpha stimulated NF-kappaB activity

alpha-MSH is an anti-inflammatory peptide which signals by binding to the melanocortin-1 receptor (MC1R) and elevating cyclic AMP in several different cells and tissues. The carboxyl terminal peptides of alpha-MSH (KPV/GKPV) are the smallest minimal sequences that prevent inflammation, but it is not known if they operate via MC1R or cyclic AMP. The aim of this study was to examine the intracellular signaling potential of the GKPV peptide sequence when immobilized to polystyrene beads via a polyethylene glycol moiety. Beads containing an immobilized GKPV peptide were investigated for their ability to inhibit proinflammatory tumor necrosis factor-alpha (TNF-alpha) stimulated activation of NF-kappaB in HBL cells stably transfected with an NF-kappaB-luciferase reporter construct. Peptide functionalized beads were compared with the ability of soluble peptide alone (alpha-MSH or GKPV) or non-functionalized beads to inhibit TNF-alpha stimulated activation of NF-kappaB. GKPV peptide functionalized beads significantly inhibited NF-kappaB-luciferase activity in comparison to beads containing no peptide moiety in one of two growths conditions investigated. Soluble alpha-MSH and GKPV peptides were also confirmed to inhibit NF-kappaB-luciferase. The present study suggests that the carboxyl terminal MSH peptide acts via a cell receptor-based mechanism and furthermore may support the potential use of such immobilized ligands for anti-inflammatory therapeutic use.     

Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction

Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Abeta in cell culture. The potentiation of toxicity is greatest for Abeta1-42 > Abeta1-40 > mouse/rat Abeta1-40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H(2)O(2) in cell-free assays and to exhibit amyloid pathology. The copper complex of Abeta1-42 has a highly positive formal reduction potential ( approximately +500-550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Abeta-mediated oxidative damage in Alzheimer's disease.     

Cytochrome c' from Paracoccus denitrificans: spectroscopic studies consistent with a role for the protein in nitric oxide metabolism

Cytochrome c' was purified from the denitrifying bacterium Paracoccus denitrificans and the interaction of the protein with nitric oxide was examined spectroscopically. Two distinct types of haem-nitrosyl electronic absorption spectrum were observed, which were dependent upon [NO]. When cytochrome c' was saturated with NO, alpha and beta bands were centred at 562 nm and 530 nm, whereas with sub-saturating concentrations of NO the alpha and beta bands were red-shifted to 578 nm and 542 nm respectively. Further spectroscopic analysis showed that purified cytochrome c', added to suspensions of P. denitrificans, is able to complex with the NO which is formed as a freely diffusible intermediate of denitrification. In the presence of added NO-3 or NO-2, 40-60% of Fe(II)-cytochrome c' forms a 6-coordinate haem-nitrosyl complex. In the absence of nitrogen oxyanions or NO whole denitrifying cells are able to remove the NO from a Fe(II)-cytochrome c'-NO complex. These findings support the hypothesis that the physiological function of this enigmatic cytochrome involves the reversible binding of nitric oxide.     

Copper mediates dityrosine cross-linking of Alzheimer's amyloid-beta

We have previously reported that amyloid Abeta, the major component of senile plaques in Alzheimer's disease (AD), binds Cu with high affinity via histidine and tyrosine residues [Atwood, C. S., et al. (1998) J. Biol. Chem. 273, 12817-12826; Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233] and produces H(2)O(2) by catalyzing the reduction of Cu(II) or Fe(III) [Huang, X., et al. (1999) Biochemistry 38, 7609-7616; Huang, X., et al. (1999) J. Biol. Chem. 274, 37111-37116]. Incubation with Cu induces the SDS-resistant oligomerization of Abeta [Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233], a feature characteristic of neurotoxic soluble Abeta extracted from the AD brain. Since residues coordinating Cu are most vulnerable to oxidation, we investigated whether modifications of these residues were responsible for Abeta cross-linking. SDS-resistant oligomerization of Abeta caused by incubation with Cu was found to induce a fluorescence signal characteristic of tyrosine cross-linking. Using ESI-MS and a dityrosine specific antibody, we confirmed that Cu(II) (at concentrations lower than that associated with amyloid plaques) induces the generation of dityrosine-cross-linked, SDS-resistant oligomers of human, but not rat, Abeta peptides. The addition of H2O2 strongly promoted Cu-induced dityrosine cross-linking of Abeta1-28, Abeta1-40, and Abeta1-42, suggesting that the oxidative coupling is initiated by interaction of H2O2 with a Cu(II) tyrosinate. The dityrosine modification is significant since it is highly resistant to proteolysis and is known to play a role in increasing structural strength. Given the elevated concentration of Cu in senile plaques, our results suggest that Cu interactions with Abeta could be responsible for causing the covalent cross-linking of Abeta in these structures.     

A calpain-like proteolytic activity produces the limited cleavage at the N-terminal regulatory domain of rabbit skeletal muscle AMP deaminase: evidence of a protective molecular mechanism

On storage at 4 degrees C, rabbit skeletal muscle AMP deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.     

Expression of functional hypodermin A,a serine protease from Hypoderma lineatum (Diptera,Oestridae), in Schneider 2 cells

Hypodermin A (HA) is a serine protease secreted by first-instar Hypoderma lineatum larvae (Oestridae, Diptera). It plays a crucial role in induced immunosuppression during hypodermosis. This report describes the production of recombinant HA in Drosophila melanogaster Schneider 2 cells, its purification and its characterization, and compares it with the protease extracted form parasite larvae. The recombinant protein and the native HA have similar biochemical and biological features. Activity of the recombinant protease on the lymphocyte proliferation inhibition and on membrane antigen cleavage was tested and shown to be similar to the native one. Tunicamycin treatment of the recombinant HA shows that the two putative glycosylation sites carry glycan residues. Unglycosylated recombinant HA has the same enzymatic activity as the fully glycosylated protein, indicating that glycosylation is not important for the protease activity of HA.