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The ancestral galectin from the sponge Geodia cydonium (GCG) is classified on a structural basis to the prototype subfamily, whereas its carbohydrate-binding specificity is related to that of the mammalian chimera-type galectin-3. This dual coordination reveals GCG as a potential precursor of the later evolved galectin subfamilies, which is reflected in the primary structure of the protein. This study provides evidence that GCG is the LECT1 gene product, while neither a previously described LECT2 gene nor a functional LECT2 gene product was found in the specimen under investigation. The electrophoretically separated protein isomers with apparent molecular masses of 13, 15, and 16 kDa correspond to variants of the LECT1 protein-exhibiting peptide sequence polymorphisms that concern critical positions of the carbohydrate recognition domain (13 kDa: Leu51, Asn55, His130, Gly137; 15 kDa: Ser51, Asn55, Asn130, Gly137; 16 kDa: Ser51, Tyr55, Asn130, Glu137). Four residues, highly conserved in the galectin family, are substituted. None of the residues claimed to be involved in interactions with GalNAc alpha 1-3 moieties at an extended binding subsite of galectin-3 was identified in the corresponding positions of GCG. Apparently, the substitutions do not confer distinct binding characteristics to the GCG variants as evidenced by binding studies with a recombinantly expressed 15-kDa isoform. The natural isoforms as well as the recombinant 15-kDa isoform oligomerize by the formation of non-covalent heteromeric or homomeric complexes. A phosphorylation of the galectin was confirmed neither by mass spectrometry nor by alkaline phosphatase treatment combined with isoelectric focusing
Photochemical and photophysical properties were investigated for poly(arylenevinylene)s containing a flexible biphenyl "hinge" unit by applying one-photon (OP) and two-photon (TP) excitation to explore excited-state properties. The poly(arylenevinylene)s were poly[(2,5-dihexyloxy-p-phenylenevinylene)-alt-(4,4'-dihexyloxy-3,3'-biph enylenevinylene)] (1), poly[(2,5-dihexyloxy-p-phenylenevinylene)-alt-(2,2'-dihexyloxy-3,3'-biph enylenevinylene)] (2), and poly[(2,5-dihexyloxy-p-phenylenevinylene)-alt-(2,2'-biphenylene-vinylene )] (3). Effective emission quantum yields and related photonic properties were evaluated on a realistic per-chromophore basis using effective conjugation lengths based on the Strickler-Berg relationship. intramolecular photocyclization was deduced to occur in the one case where the biphenyl molecular connectivity permitted the reaction, based on matrix- assisted loser desorption/ionization time-of-flight (MALDI-TOF), heteronuclear multiple-quantum coherence (HMQC)-NMR, and gel-permeation chromatography (GPC) results. The various photoprocesses could be induced by either OP or TP excitation, though the first excited singlet state is the photoactive state. The higher excitation energy 1 of the TP excited state favors indirect population of the S, state by electronic coupling between the TP and OP excited states [lambda(max)(TPE) (nm): 726; delta (GM)([9]): 1 = 229, 2 = 215, 3 = 109). Photochemical processes occurring from the lowest OP excited state (S-1) could therefore also be indirectly induced by TP excitation
Background: Transthyretin (TTR), a traditional biomarker for nutritional and inflammatory status exists in different molecular variants of yet unknown importance. A truncated form of TTR has recently been described to be part of a set of biomarkers for the diagnosis of ovarian cancer. The main aim of the study was therefore to characterize differences in microheterogeneity between ascitic fluid and plasma of women affected with ovarian cancer and to evaluate the tumor site as the possible source of TTR. Methods: Subjects were 48 women with primary invasive epithelial ovarian cancer or recurrent ovarian carcinoma. The control group consisted of 20 postmenopausal women. TTR and retinol-binding protein (RBP) levels were measured by enzyme-linked immunoassay ( ELISA) and C-reactive protein (CRP) levels by a high- sensitivity latex particle turbidimetric assay. The molecular heterogeneity of TTR was analysed using immunoprecipitation and matrix-associated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Presence of TTR in tumor tissue was determined with indirect peroxidase immunostaining. Results: TTR and RBP (mu g/ml) levels in serum were 148.5 +/- 96.7 and 22.5 +/- 14.8 in affected women compared to 363.3 +/- 105.5 and 55.8 +/- 9.3 in healthy postmenopausal women ( p < 0.01). In ascitic fluid, levels were 1.02 +/- 0.24 and 4.63 +/- 1.57 mu g/ml, respectively. The mean levels of TTR and RBP in serum showed a tendency to decrease with the severity of the disease and were lower in affected women whose CRP levels were > 40 mg/ml ( p = 0.08 for TTR; p < 0.05 for RBP). No differences in TTR microheterogeneity were observed between TTR isolated from serum of affected and healthy women or from ascitic fluid. TTR occurred rather consistently in four variants. Mass signals were at 13758 +/- 7, 13876 +/- 13 ( greatest intensity), 13924 +/- 21 and 14062 +/- 24 Da, representing native, S-cysteinylated, S-cysteinglycinylated and glutathionylated TTR, respectively. Serum of healthy and affected women as well as ascitic fluid contained the truncated fragment of TTR ( 12828 +/- 11 Da). No immunoreactive TTR was observed in the tumor sites. Conclusion: The severity of the cancer associated catabolism as well as the inflammation status affect serum TTR and RBP levels. Neither TTR nor its truncated form originates from tumor tissue and its occurrence in ascites may well reflect the filtration from blood into ascitic fluid
Mitochondrial diseases may be caused by numerous mutations that alter proteins of the respiratory chain and of other metabolic pathways in the mitochondrium. For clinicians this disease group poses a considerable diagnostic challenge due to ambiguous genotype-phenotype relationships. Until now, only 30 % of the mitochondriopathies can be diagnosed at the molecular level. We therefore need a new diagnostic tool that offers a wide view on the mitochondrial proteins. Here, we present a method to generate a high-resolution, large-gel two-dimensional gel electrophoretic (2-DE) map of a purified fraction of mitochondrial proteins from Epstein-Barr virus-immortalized lymphoblastoid cell line (LCL). LCLs can be easily obtained from patients and control subjects in a routine clinical setting. They often express the biochemical phenotype and can be cultured to high cell numbers, sufficient to gain enough purified material for 2- DE. In total we identified 166 mitochondrial proteins. Thirteen proteins were earlier not known to be of mitochondrial origin. Thirty-nine proteins were associated with human diseases ranging from respiratory chain enzyme deficiencies to disorders of P-oxidation and amino acid metabolism. This 2-DE map is intended to be the first step to diagnose mitochondrial diseases at the proteomic level