We have characterized microvillus membrane (MVM) lipid composition and derived estimates of membrane fluidity using fluorescence polarization [with 1,6-diphenyl-1,3,5-hexatriene (DPH) as the probe] in MVM from the jejunum and ileum of suckling (day 14-20) and mature postweaning (day 28-49) rats. The anisotropic values are lower (i.e., increased fluidity) in MVM from jejunum and ileum of suckling rats compared with values from MVM of mature rats, suggesting a more disordered molecular environment in MVMs of suckling animals, although the ileal MVM was relatively less fluid than jejunal membranes in both groups. Anisotropic determinations in protein-free MVM liposomes demonstrate that protein-lipid interactions are major determinants of fluidity estimates. Arrhenius plots of DPH anisotropies in MVM and MVM liposomes indicate a thermotropic lipid-phase transition at 23 degrees C in mature rat jejunum and ileum; however, no clear transition point was observed in 14- to 20-day animals. In addition, both the jejunal and ileal MVM in suckling animals contained greater amounts of total lipid, cholesterol, and phospholipid per milligram protein than did mature MVM preparations. Membrane phospholipid composition changed with age, with a decrease in sphingomyelin and an increase in phosphatidylcholine in the jejunum of the postweaning animal and with similar weight ratios of saturated to unsaturated fatty acids in both age groups. The data demonstrate that the small intestinal MVM manifests both age-dependent and regional proximal-distal differences in lipid composition and biophysical properties.(ABSTRACT TRUNCATED AT 250 WORDS)
Introduction All neoplastic cells express one or more members of a unique family of tumor-associated cell surface ubiquinone (NADH) oxidase proteins with protein disulfide-thiol interchange activity (ENOX2 or tNOX proteins) that are characteristically blocked by quinone site inhibitors with anti-cancer activity. Methods Analyses using two-dimensional gel electrophoresis with detection on western blots using a pan ENOX2 recombinant antibody revealed unique ENOX2 isoforms or unique combinations of isoforms of differing molecular weights and/or isoelectric points in sera of patients with cancers of different cellular or tissue origins. Results and Discussion Isoform presence provides for broad-range cancer detection. The specific patterns and molecular weights of the isoforms present allows for identification of the cell type and/or tissue of origin of the neoplasm. ENOX2 isoform presence and relative amounts are largely independent of stage but may be proportional to tumor burden to provide indications of response to therapy and disease progression.
BackgroundMalignant mesothelioma is an aggressive, almost uniformly fatal tumor, caused primarily by exposure to asbestos. In this study, serum presence of mesothelioma-specific protein transcript variants of ecto-nicotinamide adenine dinucleotide oxidase disulfide-thiol exchanger 2 (ENOX2), a recently identified marker of malignancy, were investigated using the ONCOblot tissue of origin cancer detection test.MethodsSequential serum samples collected from asbestos-exposed individuals prior to the development of frank mesothelioma were assayed for ENOX2 presence by 2-D gel immunoblot analysis to determine how long in advance of clinical symptoms mesothelioma-specific ENOX2 transcript variants could be detected.ResultsTwo mesothelioma-specific ENOX2 protein transcript variants were detected in the serum of asbestos-exposed individuals 4–10 years prior to clinical diagnosis of malignant mesothelioma (average 6.2 years). Either one or both ENOX2 protein transcript variants indicative of malignant mesothelioma were absent in 14 of 15 subjects diagnosed with benign pleural plaques either with or without accompanying asbestosis.ConclusionsIn a population of asbestos-exposed subjects who eventually developed malignant mesothelioma, ENOX2 protein transcript variants characteristic of malignant mesothelioma were present in serum 4–10 years in advance of clinical symptoms. As with all biomarker studies, these observations require validation in a larger, independent cohort of patients and should include prospective as well as retrospective sampling.
A proteomics approach with detection on western blots using an S‐peptide tagged pan‐tNOX (ENOX2) recombinant (scFv) antibody followed by alkaline phosphatase‐linked anti S has revealed a family of more than 20 ENOX2 isoforms of varying molecular weights (34 to 94 kDa) and mostly of low isoelectric points (4.6 ± 0.7) based on serum analysis. Different isoforms characterize cancers of different tissue origins indicative of both cancer presence and tissue site of origin. ENOX2 proteins are cancer‐associated and differ from constitutive (CNOX or ENOX1) proteins primarily by the absence of a drug binding site to which the cancer‐specific scFv is directed. All are located on the cell surface where they function both as terminal oxidases for plasma membrane electron transport and carry out protein disulfide‐thiol interchange. These proteins are shed into the blood and can also be found in urine. The tNOX isoform technology is under development as a clinical aid to identify unknown or uncertain primary cancers, evaluation of metastatic spread in post surgery patients, monitoring remission following cessation of therapy and for early diagnosis in at‐risk populations.
ENOX proteins with an oscillatory pattern of production of superoxide (measured by ferricytochrome c reduction) and with a period length of 26 min increase linearity with age beginning at about 30 y to a maximum of about age 60. The proteins are shed and appear in serum, saliva and urine. Enhanced arNOX activity correlates with age and with oxidative changes contributing to skin aging. Topical cosmetic preparations containing substances that block arNOX activity are under evaluation to reduce visible symptoms of skin aging.
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