The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding

Alpha-synuclein (αS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αS is abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length αS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the αS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu.     

Glutamatergic Activation of Hippocampal Phospholipase D: Postnatal Fading and Receptor Desensitization

Abstract: Phospholipase D (PLD) activity was determined in rat hippocampal slices between postnatal days 3 and 35. After birth, basal PLD activity was low and, within 2 weeks, increased to reach a plateau that was maintained up to the adult age. Likewise the response to glutamate developed postnatally to reach a maximum at day 8, but then faded rapidly and was almost absent at day 35. Activation of PLD by 4β-phorbol 12β,13α-dibutyrate (PDB) was independent of age, whereas the effect of aluminum fluoride (AlF₄⁻) increased to a plateau within the first week. At day 8, PLD stimulation by glutamate via metabotropic receptors involved protein kinase C activation, but was independent of Ca²⁺ influx; the time course of PLD activation by PDB or AlF₄⁻ was linear throughout the experiment, whereas the response to glutamate or 1-aminocyclopentane-1,3-dicarboxylic acid followed a biphasic pattern: the rapid "first phase activation" desensitized within a few minutes and disclosed a small, but maintained "second phase." Pretreatment experiments confirmed desensitization of PLD activation by glutamate, but not by AlF₄⁻ or PDB. The biphasic pattern of glutamatergic PLD activation changed during development, i.e., the first phase activation faded and the second phase remained. These results were fully confirmed by the time courses of the PLD-mediated efflux of choline evoked by glutamate. In conclusion, postnatal glutamatergic activation of hippocampal PLD is composed of a pronounced and desensitizing first phase activation and a small, but nondesensitizing second phase. The first, but not the second, phase activation fades rapidly during development. The hypothesis is discussed that the glutamatergic activation of PLD occurs along different pathways in neonate and adult tissue.     

γδ T-cell receptor repertoire in human peripheral blood and thymus

T-cell clones expressing the T-cell receptor (Tcr) were generated from peripheral blood lymphocytes (PBLs) and from a thymus sample. In the panel of ten thymus-derived clones, four Tcr phenotypes [as defined by the reaction of monoclonal antibodies (mAbs) directed against known V and V regions] were identified. All the clones lacked expression of the V3 V region, while seven clones were V1+ . V1 was found in combination with V9 or with undefined VVregions. In addition, two other Tcr phenotypes were identified on these clones: V9⁺ V1^– V3^– and V9^– V1^– V3^– One of the clones expressed CD4 and another was CD8positive. The remaining clones were CD4^– CD8^–. In the panel of 76 PBL-derived, Tcr-bearing clones, five Tcr phenotypes could be identified. In contrast to the thymus-derived clones, 30% of the clones were V3⁺ whereas V1 was expressed by a minority of the clones only. One clone was CD4-positive and approximately 30% of the clones were CD8-positive. Four of the five mAb-defined Tcr phenotypes could be identified on both thymus and PBL-derived T-cell clones. However, biochemical analysis of the Tcrs demonstrates differences in the usage of Ct- and C2-encoded y chains by T cells derived from the thymus and PBLs. The results therefore indicate that, at the clonal level, similarities and differences exist between the Tcr repertoires expressed in the thymus and by PBLs. Furthermore, they indicate that combinatorial Tcr heterogeneity is larger than has so far been described. The receptor diversity, combined with the potential of Tcr+ cells to express CD4 or CD8, indicates that these cells are a heterogeneous population that might mediate a number of immune functions.     

Virulence factors of Bordetella pertussis

Clearly, B. pertussis has evolved very elaborate mechanisms to maintain itself in the human host. Three different proteins (FHA, pertussis toxin and fimbriae) have been implicated in adherence. Furthermore, a number of toxins are produced (pertussis toxin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin) which destroy the clearance mechanisms of the respiratory tract, or suppress the immune response. There is evidence that B. pertussis may survive intracellularly, and the possibility that it is a facultative intracellular parasite should certainly be explored. The availability of a large number of cloned virulence genes, and a system to construct well defined mutants by allelic exchange (Stibbitz et al. 1986) will greatly facilitate the study of Bordetella virulence factors at the molecular level. It opens the possibility to construct avirulent strains, which are still able to colonize and stimulate the local immune response. Such strains may be used as live, oral vaccines, to present (heterologous) antigens to the mucosal immune system of the respiratory tract.     

Habitual Sleep Measures are Associated with Overall Body Fat,and not Specifically with Visceral Fat,in Men and Women

Objective

This study aimed to investigate the associations of sleep duration and sleep quality with visceral adipose tissue (VAT) in middle‐aged individuals.

Methods

In this cross‐sectional analysis of baseline measurements of the Netherlands Epidemiology of Obesity (NEO) study, participants underwent anthropometry and completed the Pittsburgh Sleep Quality Index (PSQI) for assessing short sleep duration (as sex‐specific age‐adjusted percentiles) and poor quality (PSQI > 5). VAT was assessed by magnetic resonance imaging in a random subgroup. We performed linear regression analyses to examine associations of short sleep and poor sleep with measures of body fat, adjusted for confounding, including total body fat in models with VAT.

Results

A total of 5,094 participants (52% women; mean age of 56 [SD 6] years), 1,947 of whom had VAT measurements, were analyzed. The difference in VAT between poor sleep (PSQI > 5) and good sleep (PSQI ≤ 5) was 7.2cm² (95% CI: 1.2‐13.8) in women and 16.1cm² (95% CI: 6.2‐26.0) in men. These differences attenuated toward the null after the adjustment for total body fat. Similar patterns of associations were observed for short sleep (lowest 10% compared with median 60%).

Conclusions

Our results suggest that measures of sleep are not specifically associated with a higher amount of VAT.     

Effects of the lunar cycle on the Galápagos fur seal,Arctocephalus galapagoensis

Summary During the 1977 and 1979 reproductive periods of the Galápagos fur seals a census taken in the mornings and evenings at Cabo Hammond, Fernandina, showed a marked, synodic lunar rhythm in numbers of animals ashore. About twice as many fur seals were ashore at full moon than at new moon. By use of two independent Fourier analysis methods, the curve of the morning counts is shown to lag 15°–20° of the lunar month behind the curve of the evening counts. The lunar effect is demonstrated for males, females, and immatures. The rhythm is also seen is demonstrated for males, females, and immatures. The rhythm is also seen in attendance data from 13 individually marked females, all but one nursing young. Reproductive events show the lunar rhythm much less markedly than do numbers ashore. This and the clear rhythm in immature numbers make it very likely that the rhythm is a year-round phenomenon, independent of reproduction.There is no reason to assume that fur seals stay on land during moonlit nights especially for social interaction. It is then hypothesized that fur seals avoid moonlight at sea. If so, the peak of numbers ashore at full moon and the negative phase angle difference of the evening curve against the morning curve can be explained with the shift, and the varying duration and brightness, of the moonlit part of the night over the lunar cycle. Two hypotheses which might account for this moonlight avoidance are discussed: (1) predator (shark) avoidance and (2) varying feeding efficiency of the fur seals due to the influence of moonlight on the vertical distribution of prey.     

THE ULTRASTRUCTURE OF THE M LINE IN SKELETAL MUSCLE

By electron microscopy, the ultrastructure of the M line was investigated in fibers from frog nonglycerinated semitendinosus muscles at body length and at different degrees of shortening and stretch. The M line appeared as a line of high electron opacity in the middle of the A band. Its framework consists of: (i) three (four or five) arrays of transverse M bridges, 200 A apart, connecting each A filament with its six neighbors; (ii) M filaments, parallel to the A filaments, passing through the M line and linking each set of M bridges together. In the shortened fiber the M line remained distinct. At high degrees of stretch, the M line became fainter or indiscernible. This appearance reflects a misalignment of the M components caused by a staggering of the A filaments. The M line reappeared after release of fibers stretched 70–80% above equilibrium length. On the basis of the structural analysis, the possible function of the M line is compared with that of the Z line, and a model is suggested for the M line.