The major constituents in grape seed and pine bark extracts are proanthocyanidins. To evaluate material available to consumers, select lots were analyzed using high-performance liquid chromatography, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), gel permeation chromatography (GPC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Atmospheric pressure chemical ionization (APCI) LC/MS was used to identify monomers, dimers, and trimers present. GC/MS analyses led to the identification of ethyl esters of hexadecanoic acid, linoleic acid, and oleic acid, as well as smaller phenolic and terpene components. The GPC molecular weight (MW) distribution indicated components ranging from approximately 162 to approximately 5500 MW (pine bark less than 1180 MW and grape seed approximately 1180 to approximately 5000 MW). MALDI-TOF MS analyses showed that pine bark did not contain oligomers with odd numbers of gallate units and grape seed contained oligomers with both odd and even numbers of gallate. Reflectron MALDI-TOF MS identified oligomers up to a pentamer and heptamer, and linear MALDI-TOF MS showed a mass range nearly double that of reflectron analyses.

Oxygen toxicity is one of the major risk factors in the development of the chronic lung disease or bronchopulmonary dysplasia in premature infants. Using proteomic analysis, we discovered that mitochondrial aldehyde dehydrogenase (mtALDH or ALDH2) was downregulated in neonatal rat lung after hyperoxic exposure. To study the role of mtALDH in hyperoxic lung injury, we overexpressed mtALDH in human lung epithelial cells (A549) and found that mtALDH significantly reduced hyperoxia-induced cell death. Compared with control cells (Neo-A549), the necrotic cell death in mtALDH-overexpressing cells (mtALDH-A549) decreased from 25.3 to 6.5%, 50.5 to 9.1%, and 52.4 to 15.1% after 24-, 48-, and 72-h hyperoxic exposure, respectively. The levels of intracellular and mitochondria-derived reactive oxygen species (ROS) in mtALDH-A549 cells after hyperoxic exposure were significantly lowered compared with Neo-A549 cells. mtALDH overexpression significantly stimulated extracellular signal-regulated kinase (ERK) phosphorylation under normoxic and hyperoxic conditions. Inhibition of ERK phosphorylation partially eliminated the protective effect of mtALDH in hyperoxia-induced cell death, suggesting ERK activation by mtALDH conferred cellular resistance to hyperoxia. mtALDH overexpression augmented Akt phosphorylation and maintained the total Akt level in mtALDH-A549 cells under normoxic and hyperoxic conditions. Inhibition of phosphatidylinositol 3-kinase (PI3K) activation by LY294002 in mtALDH-A549 cells significantly increased necrotic cell death after hyperoxic exposure, indicating that PI3K-Akt activation by mtALDH played an important role in cell survival after hyperoxia. Taken together, these data demonstrate that mtALDH overexpression attenuates hyperoxia-induced cell death in lung epithelial cells through reduction of ROS, activation of ERK/MAPK, and PI3K-Akt cell survival signaling pathways.

Since little is known regarding osteocytes, cells embedded within the mineralized bone matrix, a proteomics approach was used to discover proteins more highly expressed in osteocytes than in osteoblasts to determine osteocyte specific function. Two proteomic profiles obtained by two different proteomic approaches using total cell lysates from the osteocyte cell line MLO-Y4 and the osteoblast cell line MC3T3 revealed unique differences. Three protein clusters, one related to glycolysis, (Phosphoglycerate kinase 1, fructose-bisphosphate aldolase A, hypoxia up-regulated 1 [ORP150], triosephosphate isomerase), one to protein folding (Mitochondrial Stress-70 protein, ORP150, Endoplasmin), and one to actin cytoskeleton regulation (Macrophage-capping protein [CapG], destrin, forms of lamin A and vimentin) were identified. Higher protein expression of ORP-150, Cap G, and destrin in MLO-Y4 cells compared to MC3T3 cells was validated by gene expression, Western blotting, and in vivo expression. These proteins were shown to be selective in osteocytes in vivo using immuno-staining of mouse ulnae. Destrin was most highly expressed in embedding osteoid osteocytes, GapG in embedded osteocytes, and ORP150 in deeply embedded osteocytes. In summary, the proteomic approach has yielded important information regarding molecular mechanisms used by osteocytes for embedding in matrix, the formation of dendritic processes, and protection within a hypoxic environment.

ABSTRACT:The objective was to study the photocationic polymerization of an expanding monomer, 1,5,7,11-tetraoxaspiro[5.5]undecane (TOSU), and an aromatic dioxirane, bisphenol A diglycidyl ether (BADGE). Both homopolymerizations and binary polymerizations were conducted. The homopolymer, poly(TOSU), was found to be a linear poly(carbonate), which was soluble in acetone. Poly-(BADGE) products contained ether linkages in addition to primary and secondary alcohol functionalities. Binary polymerization products varied depending on the irradiation time and length of dark cure.13 C-NMR analysis of binary polymerizate products revealed peaks not seen in homopolymer spectra consistent with the formation of copolymer linkages. Mass spectrometry data revealed peaks consistent with oligomers that contained both TOSU and BADGE mer units. The structures of key reaction products were proposed.