Cutting Edge: Hormonal milieu, not antigenic specificity, determines the mature phenotype of autoreactive B cells

Although both marginal zone and follicular B cells produce anti-DNA Abs in murine models of systemic lupus erythematosus, it has been unclear whether these distinct B cell subsets make identical or different Abs. Single-cell analysis demonstrates that the same DNA-reactive B cells can mature to either subset, depending on the hormonal environment. Anti-DNA B cells in estradiol-treated mice become marginal zone cells while identical cells from prolactin-treated mice become follicular cells. The B cell receptor signaling pathway is influenced by hormonal milieu. Thus, hormonal milieu and perhaps B cell receptor signaling, but not antigenic specificity, correlates with the differentiation pathway. These observations have implications for the pathogenesis and treatment of autoimmune disease.     

Dynamic behavior of fatty acid spin labels within a binding site of soybean lipoxygenase-1

The putative substrate-binding site in lipoxygenases is long and internal. There is little direct evidence about how the unsaturated fatty acid substrates enter and move within the cavity to position themselves correctly for electron transfer reactions with the catalytic non-heme iron. An EPR spectroscopy approach, with spin-labeled fatty acids, is taken here to investigate dynamic behavior of fatty acids bound to soybean lipoxygenase-1. The probes are labeled on C5, C8, C10, C12, and C16 of stearic acid. The EPR-determined affinity for the enzyme increases as the length of the alkyl end of the probe increases, with a DeltaDeltaG of -190 cal/methylene. The probes in the series exhibit similar enhanced paramagnetic relaxation by the iron center. These results indicate that the members of the series have a common binding site. All of the bound probes undergo considerable local mobility. The stearate spin-labeled at C5 has the highest affinity for the lipoxygenase, and it is a competitive inhibitor, with a K(i) of 9 muM. Surprisingly, this stearate labeled near the carboxyl end undergoes more local motion than those labeled in the middle of the chain, when it is bound. This shows that the carboxyl end of the fatty-acid spin label is not rigidly docked on the protein. During catalysis, repositioning of the substrate carboxyl on the protein surface may be coupled to motion of portions of the chain undergoing reaction.     

Probing the production of amidated peptides following genetic and dietary copper manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.     

Familial Aggregation between the 14th and 21st Century and Type 2 Diabetes Risk in an Isolated Dutch Population

IntroductionThe development of type 2 diabetes results from an interaction of hereditary factors and environmental factors. This study aimed to investigate the contribution of interrelatedness to the risk of developing type 2 diabetes in an isolated Dutch population.ResultsPatients with type 2 diabetes were more interrelated, expressed by a higher KC compared to controls (7.2 vs. 5.2, p=0.001). First, second and third degree relatives had an increased risk of developing type 2 diabetes. Second degree relatives had a similar risk,1.7 (1.5-2.0) as third degree relatives,1.8 (1.5-2.2). Spouses of patients with diabetes had a 3.4 (2.7-4.4) higher risk of developing type 2 diabetes.ConclusionsInterrelatedness was higher among inhabitants with type 2 diabetes compared to controls. This differences extended beyond the nuclear family, thereby supporting the hypothesis that interrelatedness contributed to the development of type 2 diabetes on Urk. However, the size of this effect was small and the patterns of risk in first, second and third degree relatives suggested that factors other than interrelatedness were the main contributors to the development of type 2 diabetes on Urk.     

Interplay between structure and dynamics in chitosan films investigated with solid-state NMR, dynamic mechanical analysis, and X-ray diffraction

Modern solid-state NMR techniques, combined with X-ray diffraction, revealed the molecular origin of the difference in mechanical properties of self-associated chitosan films. Films cast from acidic aqueous solutions were compared before and after neutralization, and the role of the counterion (acetate vs Cl(-)) was investigated. There is a competition between local structure and long-range order. Hydrogen bonding gives good mechanical strength to neutralized films, which lack long-range organization. The long-range structure is better defined in films cast from acidic solutions in which strong electrostatic interactions cause rotational distortion around the chitosan chains. Plasticization by acetate counterions enhances long-range molecular organization and film flexibility. In contrast, Cl(-) counterions act as a defect and impair the long-range organization by immobilizing hydration water. Molecular motion and proton exchange are restricted, resulting in brittle films despite the high moisture content.     

Ancient and recent evolutionary history of the bruchid beetle, Acanthoscelides obtectus Say, a cosmopolitan pest of beans

Acanthoscelides obtectus Say is a bruchid species of Neotropical origin, and is specialized on beans of the Phaseolus vulgaris L. group. Since the domestication and diffusion of beans, A. obtectus has become cosmopolitan through human-mediated migrations and is now a major pest in bean granaries. Using phylogeographic methods applied to mitochondrial DNA (mtDNA) and nuclear microsatellite molecular markers, we show that the origin of this species is probably further south than Mesoamerica, as commonly thought. Our results also indicate that A. obtectus and its Mesoamerican sister species Acanthoscelides obvelatus, two morphologically close species differing principally in voltinism, speciated in allopatry: A. obtectus (multivoltine) arising in Andean America and A. obvelatus (univoltine) in Mesoamerica. In contrast to Mesoamerica where beans fruit once yearly, wild beans in Andean America fruit year-round, especially in regions showing little or no seasonality. In such habitats where resources are continuously present, multivoltinism is adaptive. According to existing hypotheses, multivoltinism in A. obtectus is a new adaptation that evolved after bean domestication. Our data suggest the alternative hypothesis that multivoltinism is an older trait, adapted to exploit the year-round fruiting of wild beans in relatively aseasonal habitats, and allowed A. obtectus to become a pest in bean granaries. This trait also permitted this species to disperse through human-mediated migrations associated with diffusion of domesticated beans. We also show that diversity of Old World A. obtectus populations can be quite well explained by a single colonization event about 500 bp. Human-mediated migrations appear not to be rare, as our results indicate a second more recent migration event from Andean America to Mexico.     

Fabrication of Sub-10-nm Plasmonic Gaps for Ultra-Sensitive Raman Spectroscopy

Plasmonics - The past two decades have witnessed the explosion of activities in the field of surface enhanced Raman spectroscopy (SERS). SERS platforms employ nano-structures that excite plasmonic...