The effects of normal saline instillation in conjunction with negative pressure wound therapy on wound healing in a porcine model.

Unlabelled: Acute and chronic wounds impact the lives of millions of patients. Since its introduction, negative pressure wound therapy using reticulated open cell foam (NPWT/ROCF) has significantly improved the healing outcome for many of these wounds.

Methods: The effects of intermittent instillation of normal saline in conjunction with NPWT were investigated to determine if instillation therapy provides additional benefits in wound healing.

O-Glycosylated 24 kDa human growth hormone has a mucin-like biantennary disialylated tetrasaccharide attached at Thr-60.

MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O-linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI-TOF/MS and ESI-MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high-performance anion-exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N-acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc(2), N-acetyl galactosamine(1), Gal(1)).

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid.

Characterization of Pseudomonas chlororaphis myovirus 201varphi2-1 via genomic sequencing, mass spectrometry, and electron microscopy.

Pseudomonas chlororaphis phage 201varphi2-1 is a relative of Pseudomonas aeruginosa myovirus phiKZ. Phage 201 phi2-1 was examined by complete genomic sequencing (316,674 bp), by a comprehensive mass spectrometry survey of its virion proteins and by electron microscopy. Seventy-six proteins, of which at least 69 have homologues in phiKZ, were identified by mass spectrometry.

Complete genomic sequence and mass spectrometric analysis of highly diverse, atypical Bacillus thuringiensis phage 0305phi8-36.

To investigate the apparent genomic complexity of long-genome bacteriophages, we have sequenced the 218,948-bp genome (6479-bp terminal repeat), and identified the virion proteins (55), of Bacillus thuringiensis bacteriophage 0305phi8-36. Phage 0305phi8-36 is an atypical myovirus with three large curly tail fibers. An accurate mode of DNA pyrosequencing was used to sequence the genome and mass spectrometry was used to accomplish the comprehensive virion protein survey.

Tryptophan 334 oxidation in bovine cytochrome c oxidase subunit I involves free radical migration.

A single tryptophan (W(334(I))) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W(334(I)) is converted to hydroxytryptophan as identified by reversed-phase HPLC-electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS-PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively.

Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH.

A yeast mutant lacking the two major cytosolic sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1p) and NADP+-specific isocitrate dehydrogenase (Idp2p), has been demonstrated to lose viability when shifted to medium with acetate or oleate as the carbon source. This loss in viability was found to correlate with an accumulation of endogenous oxidative by-products of respiration and peroxisomal beta-oxidation. To assess effects on cellular protein of endogenous versus exogenous oxidative stress, a proteomics approach was used to compare disulfide bond-containing proteins in the idp2Deltazwf1Delta strain following shifts to acetate and oleate media with those in the parental strain following similar shifts to media containing hydrogen peroxide.

Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS.

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means.

Identification of phosphorylation sites in insulin receptor substrate-1 by hypothesis-driven high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) can regulate tyrosine phosphorylation of IRS-1 and subsequent insulin signaling. The 182 serine and 60 threonine residues in IRS-1 make position-by-position analysis of potential phosphorylation sites by mutagenesis difficult. Tandem mass spectrometry provides a more efficient way to identify phosphorylated residues in IRS-1.

Phosphorylation of Grb10 by mitogen-activated protein kinase: identification of Ser150 and Ser476 of human Grb10zeta as major phosphorylation sites.

Grb10 is a Src-homology 2 (SH2) and Pleckstrin-homology (PH) domain-containing protein that binds to several autophosphorylated receptor tyrosine kinases including the insulin receptor (IR). Our previous studies showed that Grb10 underwent insulin-stimulated serine phosphorylation, yet the kinase(s) responsible for phosphorylation and the sites of the phosphorylation remain unknown. In this report, we show that Grb10 is a direct substrate of the p42/44 mitogen-activated protein kinase (MAPK).

Early N-terminal changes and caspase-6 cleavage of tau in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive amnestic dementia that involves post-translational hyperphosphorylation, enzymatic cleavage, and conformational alterations of the microtubule-associated protein tau. The truncation state of tau influences many of its pathologic characteristics, including its ability to assume AD-related conformations and to assemble into filaments. Cleavage also appears to be an important marker in AD progression.

Proteomic identification of specific oxidized proteins in ApoE-knockout mice: relevance to Alzheimer's disease.

We have examined oxidized proteins in the brain regions of wild-type (WT) and ApoE-knockout (KO) animals. Total protein oxidation in the hippocampus of young-KO (6 month) animals was approximately 2-fold greater than that of young-WT (6 month) animals and was similar to that of old-WT (18 month) and old-KO (18 month) animals. In the cortex of the same animals, the levels of total protein oxidation in all four groups were not significantly different.

Analysis of proteins stained by Alexa dyes.

Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for performing this determination.

Specific modification of two tryptophans within the nuclear-encoded subunits of bovine cytochrome c oxidase by hydrogen peroxide.

Hydrogen peroxide does more than react with the binuclear center of oxidized bovine cytochrome c oxidase and generate the well-characterized "peroxy" and "ferryl" forms. Hydrogen peroxide also inactivates detergent-solubilized cytochrome c oxidase in a time- and concentration-dependent manner. There is a 70-80% decrease of electron-transport activity, peroxidation of bound cardiolipin, modification of two nuclear-encoded subunits (IV and VIIc), and dissociation of approximately 60% of subunits VIa and VIIa.

Anti-apoptotic proteins are oxidized by Abeta25-35 in Alzheimer's fibroblasts.

We have examined the effects of the beta-amyloid peptide (Abeta(25-35)) on fibroblasts derived from subjects with Alzheimer's disease (AD) and from age-matched controls. The peptide was significantly more cytotoxic to the AD-derived fibroblasts. The level of protein oxidation was also greater in the cells from AD subjects.

Vitamin E prevents oxidation of antiapoptotic proteins in neuronal cells.

Oxidative damage to neuronal proteins appears to be central to the toxicity associated with a number of neuropathologies, including Alzheimer's disease. We have examined this by using oxidative stress to induce apoptosis in a mouse hippocampal neuronal cell line (HT-22). Oxidatively modified proteins were measured by high-resolution two-dimensional gel electrophoresis coupled with oxidation-specific immunostains.

Identification of bovine heart cytochrome c oxidase subunits modified by the lipid peroxidation product 4-hydroxy-2-nonenal.

Bovine heart cytochrome c oxidase (CcO) was inactivated by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in a time- and concentration-dependent manner with pseudo-first-order kinetics. Cytochrome c oxidase electron transport activity decreased by as much as 50% when the enzyme was incubated for 2 h at room temperature with excess HNE (300-500 microM). HNE-modified CcO subunits were identified by two mass spectrometric methods: electrospray ionization mass spectrometry (ESI/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS).