Retinal proteomic changes following unilateral optic nerve transection and early experimental glaucoma in non-human primate eyes.

In this work we compared proteomic changes in non-human primate (NHP) retinas at the onset of early experimental glaucoma (EEG) and 3 weeks after optic nerve transection (ONT), as a means to identify regulators in the retina's response to EEG and ONT. Both eyes of 7 NHPs with either unilateral EEG (n = 4) or ONT (n = 3) were enucleated. Proteins were analyzed by a label-free quantitative mass spectrometry system and the abundance of identified retinal proteins was compared between the treated eye and its contralateral control for each NHP. Cellular processes associated with regulated proteins were identified using the MetaCore program. As a result, a total of 209 and 200 proteins were identified in EEG and ONT retinas, respectively. The EEG eyes exhibited two distinguishable levels of maximum IOP: the highest IOP <27 mmHg (n = 2) and >45 mmHg (n = 2), termed mild IOP EEG and high IOP EEG eyes, respectively. A limited overlap of proteins regulated in the same direction was seen between the high IOP EEG and either the mild IOP EEG eyes or ONT eyes. Most of the proteins that were up regulated in the high IOP EEG eyes were down regulated in the mild IOP EEG eyes; the latter showed an overall down regulation that was not seen in the other two conditions. An association with cytoskeleton regulation was recognized for up-regulated proteins in the high IOP EEG eyes. We conclude that mild IOP EEG, high IOP EEG and ONT retinas exhibited condition-specific proteomic changes with little overlap between conditions. Cytoarchitecture regulation appears to be a component of the early retinal response to chronic experimental IOP elevation.

Elevated nonesterified fatty acid concentrations in severe preeclampsia shift the isoelectric characteristics of plasma albumin.

We previously hypothesized that the endothelial cell dysfunction observed in women with preeclampsia might be caused by an imbalance between circulating very low density lipoproteins and a cytoprotective pI 5.6 isoform of albumin, referred to as toxicity preventing albumin (TxPA). An accurate simplified method was developed to quantify TxPA in small volumes of pregnancy plasma by gel electrofocusing. This assay revealed that circulating TxPA concentrations in women with severe preeclampsia were significantly reduced compared to those in normal pregnant women and women with benign transient hypertension of pregnancy. Nonesterified fatty acids (NEFA) and triglycerides were elevated in plasma from women with severe preeclampsia compared to those in plasma from the two control groups. The inverse correlation between TxPA and NEFA values led us to analyze the NEFA bound to plasma albumin. Gas chromatography and mass spectrometry demonstrated no qualitative differences in the specific fatty acids bound to plasma albumin in severe preeclamptic and normal pregnant women. However, the quantity of NEFA bound to albumin was greater in preeclampsia plasma (2.5 mol NEFA/mol albumin) compared to that in normal pregnancy plasma (0.8 mol NEFA/mol albumin), accounting for the acidic pI shift observed in albumin from the former patients. Functional assays demonstrated that human very low density lipoprotein particles were toxic to human umbilical vein endothelial cells in vitro, but this toxicity was prevented by the addition of TxPA albumin to the culture medium.

Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests.

Fast-atom bombardment mass spectrometry was used to study disulfide bonding patterns in heat-denatured human recombinant macrophage colony stimulating factor (rhM-CSF). The heat-denaturated protein was studied by analysis of the pattern of peptides in the proteolytic digests. Native rhM-CSF is a homodimer with intramolecular disulfide linkages between Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146 and intermolecular linkages between Cys31-Cys31, and the pairs Cys157 and Cys159. Brief heating for 1 min leads to partial disulfide bond scrambling. In addition to the native disulfide bonds between Cys7-Cys90, Cys48-Cys139, and Cys31-Cys31, nonnative disulfide bonds were detected between Cys48-Cys90 and Cys48-Cys102. When heated for 5 min the disulfide bonds of rhM-CSF are completely scrambled and lead to nonnative intramolecular disulfide bonds between Cys48-Cys102 and Cys90-Cys102 and one intermolecular disulfide bond between Cys102-Cys102.

Transient loss of serum protective activity following short-term stress: a possible biochemical link between stress and atherosclerosis.

Very low density lipoproteins (VLDL) are toxic to aortic endothelial cells in vitro, and toxicity preventing activity (TxPA) inhibits this toxic effect of VLDL. Stress, an established arteriosclerosis risk factor, was examined for its effect on TxPA and on the ability of serum to protect endothelial cells from in vitro injury by VLDL. A standardized mirror tracing task with noise was administered to four healthy subjects. Blood samples were obtained at 0, 30, (stressor) 35, 50 and 80 min. Cortisol and non-esterified fatty acids increased during the stress period. TxPA significantly decreased following the stressor and had recovered by 80 min. When the ratio of non-TxPA/TxPA rose above 2, serum was no longer able to protect the cells from VLDL injury. If endothelial cells in vivo respond similarly to the endothelial cells in culture, the effect of stress on atherosclerosis may be mediated through these transient decreases in TxPA.

Disulfide linkages in the in vitro refolded intermediates of recombinant human macrophage-colony-stimulating factor: analysis of the sulfhydryl alkylation of free cysteine residues by fast-atom bombardment mass spectrometry.

Fast-atom bombardment mass spectrometry was used to follow the time course of disulfide bond formation during in vitro refolding of recombinant human macrophage-colony-stimulating factor. The content of iodoacetamide-alkylated half-cystines in proteolytic peptides of trapped refolding intermediates collected at 0, 6, 17, 24, and 72 hr was determined under reducing conditions. Size-exclusion high-performance liquid chromatography analyses of the collected alkylated samples indicate that aggregated monomer proceeded through a nonaggregated monomer to an intermediate dimer and finally to the fully folded and active dimer. Underalkylation was first detected by fast-atom bombardment mass spectrometry in 17-hr samples at Cys157 and Cys159 and this corresponded to the first sample containing dimer. Analyses of intermediates from subsequent time points indicated a decrease in alkylated sulfhydryls, and at 72 hr no alkylated peptide was detected. Early samples containing only monomer showed no evidence of disulfide bonds, and the occurrence of disulfide shuffling at the monomer stage could be ruled out under the highly reducing conditions used for refolding. Biological activity was not detectable in early samples but increased to 3.6% after 24 hr of refolding and to 86% of maximum at the 72-hr time point.

Which plasma factors bring about disturbance of endothelial function in pre-eclampsia?

The characteristic pathophysiological changes in pre-eclampsia are thought to be related to abnormalities of the maternal vascular endothelium. We suggest that the blood components that affect the risk of such damage are very-low-density lipoproteins (VLDL), which injure the endothelium, and toxicity-preventing activity (the pl 5.6 form of plasma albumin), which protects against VLDL-induced injury.

Assignment of the inter- and intramolecular disulfide linkages in recombinant human macrophage colony stimulating factor using fast atom bombardment mass spectrometry.

The disulfide bridges in recombinant human macrophage colony stimulating factor (rhM-CSF), a 49-kDa homodimeric protein, were assigned. The 18 cysteines in the dimer form three intermolecular and two sets of three intramolecular disulfide bonds. The intermolecular disulfide bridges hold the dimer together and form symmetric bonds in which Cys31 and Cys157/Cys159 from one monomer unit are linked to the corresponding cysteines of the second monomer. The intramolecular disulfide bonds are located between Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146, respectively. The resistance of native M-CSF to proteolytic cleavage was overcome by an initial chemical cleavage reaction using BrCN. The close proximity of four cysteines (Cys139, Cys146, Cys157, and Cys159) results in a tight core complex that makes the protein undigestable for most proteases. Digestion using endoprotease Asp-N resulted in cleavage at Asp156 near the C-terminal end of this region, thereby opening the complex structure.

Determination of the glycosylation patterns, disulfide linkages, and protein heterogeneities of baculovirus-expressed mouse interleukin-3 by mass spectrometry.

The primary structure of mouse interleukin-3 (IL-3) expressed by recombinant baculovirus-infected silkworm (Bombyx mori) larvae was analyzed by subjecting isolated IL-3 derived peptides to liquid secondary ion mass spectrometry. Two species of IL-3 were isolated from the silkworm hemolymph by reverse-phase high-pressure liquid chromatography. The major component has M(r)20-22 x 10(3) as determined by SDS-PAGE. Liquid secondary ion mass spectrometric analysis was carried out on the reduced tryptic and endopeptidase lysyl-C peptides of glycosylated and deglycosylated IL-3. These studies provided evidence that (1) Asn-16 is heterogeneously glycosylated with four different oligosaccharides, (2) Asn-86 is either nonglycosylated or has attached to it one oligosaccharide, (3) the N-glycosylation sites Asn-44 and Asn-51 are not glycosylated, and (4) there is no O-glycosylation. Liquid secondary ion mass spectrometric analysis of the unreduced tryptic peptides provided evidence for disulfide linkages between Cys-140 and Cys-79 or Cys-80 and between Cys-17 and Cys-79 or Cys-80. In comparison to the major component, a minor IL-3 species (M(r) 17-19 x 10(3) by SDS-PAGE) isolated from the hemolymph showed no difference with respect to the glycosylation pattern or the disulfide linkages, but it was cleaved between Ala-127 and Ser-128, and only a disulfide linkage between Cys-140 and Cys-79 or Cys-80 held the molecule together.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of stress and blood type on cortisol and VLDL toxicity preventing activity.

Past research has associated ABO blood type and mental stress with cardiovascular risk. We studied the effects of blood type (A vs. O) coupled with a mirror drawing stressor on very low density lipoprotein toxicity-preventing activity (TxPA) and plasma cortisol levels. Exposure to the stressor significantly decreased TxPA and increased cortisol for the total group of 25 older adult males. However, the stress response patterns of the 15 blood type A males were different from those of the 10 type O subjects. The blood type A group had higher initial levels of TxPA and cortisol as well as quicker stress recovery rates than the type O group. ABO blood type may be an important behavioral hematologic variable to assess in studies concerning biochemical stress response or cardiovascular risk.

Liquid secondary ion mass spectrometry analysis of permethylated, n-hexylamine derivatized oligosaccharides. Application to baculovirus expressed mouse interleukin-3.

Reductive amination with n-hexylamine followed by permethylation was used as a procedure for the liquid secondary ion mass spectrometry (LSIMS) analysis of Asn-linked oligosaccharides. Initial experiments with this procedure were performed on maltoheptaose. These experiments show that exhaustive methylation at the newly formed secondary nitrogen forms a quaternary ammonium salt. When this is subjected to positive ion LSIMS, an abundant M(+) ion is observed. This procedure was applied to the Asn-linked oligosaccharides released from human transferrin and ribonuclease-B. The reductively aminated, permethylated mixture of oligosaccharides from ribonuclease-B afforded a positive ion LSI mass spectrum in which M(+) ions for Mans5-9GlcNAc2 could be assigned. The positive ion LSI mass spectrum obtained from the mixture of oligosaccharides isolated from human transferrin showed M(+) ions that could be assigned to both monosialylated and disialylated biantennary complex type oligosaccharides. Reductive amination followed by permethylation of the Asn-linked oligosaccharides isolated from baculovirus expressed mouse interleukin-3 produced in Bombyx mori gave a positive ion LSI mass spectrum in which the oligosaccharides could be assigned the monosaccharide composition Man2-4[Fuc]GlcNAc2 and Man2GlcNAc2. These are believed to be dimannose, trimannose, and tetramannose chitobiose core oligosaccharides, three of which are fucosylated.

Relationship between blood groups and behavior patterns in men who have had myocardial infarction.

Consistent correlations have been found between physical dysfunctional states and blood factors. Some of these disorders have possible psychosomatic components (eg, duodenal ulcer, myocardial infarction). This study focused on the relationship between blood types and various indices of behavior patterns (eg, type A behavior scores, anger ratings) in young patients who had had an initial myocardial infarction. Patients with blood type O scored significantly higher on type A behavior scales and related indices than those having blood type A. Those with blood group B responded on several scales between those with types A and O. We discuss the utility of blood groupings in future research in the prediction of myocardial infarction, methodologic limitations, the relationship of these results to temperament studies, Jenkins Activity Survey subtest patterns, anti-H reactivity pattern, and hypotheses relating blood factors and behavioral traits in patients with psychosomatic disorders.

Aspirin improves the patency rate of seeded vena cava grafts.

The purpose of this study was to evaluate the effectiveness of aspirin (ASA) and porcine endothelial cell seeding in improving the patency rate of vena cava grafts. Thirty-nine dogs underwent infrarenal vena cava replacement by 10 cm lengths of 8 mm I.D. ringed polytetrafluoroethylene grafts. Thirty-one grafts were seeded with 1-1.5 x 10(6) porcine aortic endothelial cells while eight were not (GIII). Of the seeded group, 16 animals received no ASA (GI), while 15 others (GII) were given ASA (325 mg) daily starting two days preoperatively and continuing until sacrifice. Venograms were performed on the fourth postoperative day. Grafts were harvested 32 days after insertion and evaluated for patency rate and endothelialized surfaces. The 32-day patency rate was significantly higher for GII than for GI and III animals (67% vs. 13 and 25% respectively). Endothelialized surface was higher in GII than Gi and III (67% vs. 16% and 18% respectively). We conclude that endothelial cell seeding alone does not prevent graft closure and that a combination of ASA and cell seeding significantly increases the patency rate of vena cava grafts.

Evidence for radical anion formation during liquid secondary ion mass spectrometry analysis of oligonucleotides and synthetic oligomeric analogues: a deconvolution algorithm for molecular ion region clusters.

It is shown that one-electron reduction is a common process that occurs in negative ion liquid secondary ion mass spectrometry (LSIMS) of oligonucleotides and synthetic oligonucleosides and that this process is in competition with proton loss. Deconvolution of the molecular anion cluster reveals contributions from (M-2H).-, (M-H)-, M.-, and (M + H)-. A model based on these ionic species gives excellent agreement with the experimental data. A correlation between the concentration of species arising via one-electron reduction [M.- and (M + H)-] and the electron affinity of the matrix has been demonstrated. The relative intensity of M.- is mass-dependent; this is rationalized on the basis of base-stacking. Base sequence ion formation is theorized to arise from M.- radical anion among other possible pathways.

Effect of preclotting and precoating agents on seeded venous grafts.

This study evaluated the effect of whole blood preclotting (WB), fibronectin (FB) and "Cell Tak" (CT) precoating on the patency rate of seeded vena cava grafts. Ten cm x 8 mm ID ringed PTFE grafts were implanted in the vena-cavae of 29 dogs. Aspirin (325 mg po qd) was administered pre-operatively and continued post-operatively. After preclotting or precoating, all grafts were seeded with porcine endothelial cells. The 32 day patency rates were: 67%, 67% and 37% for WB, FB and CT, respectively (p less than 0.5 CT vs FB, WB). Endothelialized surfaces ranged from 67 +/- 35, 43 +/- 41 to 28 +/- 45% respectively for WB, FB and CT (p less than 0.05 CT vs WB). Preclotting time was shortest for WB technique. We conclude that: 1) FB and WB grafts have the highest patency rate with WB preclotting being a more cost effective and less time consuming technique; 2) endothelial seeding and Aspirin may make vena cava grafting possible.

Purification and identification of very low density lipoprotein toxicity preventing activity.

Toxicity preventing activity (TxPA) is a recently identified substance in serum which counteracts the toxic effect of very low density lipoproteins upon endothelial cells in vitro. In two clinical studies, TxPA was low in individuals with angiographically demonstrable coronary artery disease. An atherogenic index which combines TxPA with lipoprotein cholesterol values classifies individuals with coronary artery disease with an accuracy of greater than 93%. TxPA precipitates with 0.15 M trichloroacetic acid and above 3 M (NH4)2SO4. Activity is present in Cohn fractions IV4 and V and is stabilized by antioxidants. TxPA co-elutes with the albumin peak on gel filtration chromatography and as a subcomponent of albumin on ion-exchange chromatography. Isoelectric focusing resolves albumin into two major peaks with pI values of 4.8 and 5.6. The TxPA is identified as the pI 5.6 albumin peak.

Relationship of molecular radical anion abundance and calculated lowest unoccupied molecular orbital energies for polychlorinated dibenzofurans and dibenzodioxins in electron capture negative ion mass spectrometry: evidence for negative metastable ions.

Relationships were found between experimentally measured molecular radical anion abundances and calculated lowest unoccupied molecular orbital energies (epsilon LUMO) for polychlorodibenzofurans and polychlorodibenzo-p-dioxins. Anion abundances were measured using standard mass spectrometric techniques, while epsilon LUMO were calculated by the 'Complete Neglect of Differential Overlap' method. Polychlorodibenzofurans with calculated epsilon LUMO greater than or equal to 1.6 eV show 0% molecular radical anion and those with epsilon LUMO less than or equal to 1.4 eV show greater than or equal to 80% molecular radical anion abundance. Similarly, the molecular radical anion is absent for polychlorodibenzo-p-dioxins with calculated epsilon LUMO greater than or equal to 2.0 eV. A trend towards greater molecular radical ion relative abundance appears for 2.0 eV greater than or equal to epsilon LUMO greater than or equal to 1.0 eV and a maximum is reached around 1 eV, whereupon the molecular ion abundance diminishes with lower epsilon LUMO. B/E linked scan analysis indicates that chlorodioxins with epsilon LUMO less than 1 eV give increasing amounts of metastable anions.

Coronary disease prediction using a new atherogenic index.

This report demonstrates the utilization of a new serum factor, Toxicity Preventing Activity (TxPA) in the diagnosis of coronary disease prone individuals. Our laboratory has recently identified TxPA, which offsets the toxicity of very low density lipoproteins (VLDL) upon arterial cells in vitro. In the present study, we measured TxPA activity and serum lipoprotein levels in 73 individuals undergoing coronary angiography. Serum from control subjects demonstrated 270% more TxPA than aged matched individuals with angiographically demonstrable coronary disease (CHD). When TxPA was combined with serum lipoprotein values, a new atherogenic index was generated which further distinguished these individuals with CHD from non-angiographed controls. These results demonstrate that TxPA is a new protective factor in coronary artery disease, and that the new atherogenic index provides for the first time an accurate classification of individuals with coronary artery disease.