Insulin at pH 2: structural analysis of the conditions promoting insulin fibre formation

When insulin solutions are subjected to acid, heat and agitation, the normal pattern of insulin assembly (dimers-->tetramers-->hexamers) is disrupted; the molecule undergoes conformational changes allowing it to follow an alternative aggregation pathway (via a monomeric species) leading to the formation of insoluble amyloid fibres. To investigate the effect of acid pH on the conformation and aggregation state of the protein, the crystal structure of human insulin at pH 2.1 has been determined to 1.6 A resolution. The structure reveals that the native fold is maintained at low pH, and that the molecule is still capable of forming dimers similar to those found in hexameric insulin structures at higher pH. Sulphate ions are incorporated into the molecule and the crystal lattice where they neutralise positive charges on the protein, stabilising its structure and facilitating crystallisation. The sulphate interactions are associated with local deformations in the protein, which may indicate that the structure is more plastic at low pH. Transmission electron microscopy analysis of insulin fibres reveals that the appearance of the fibres is greatly influenced by the type of acid employed. Sulphuric acid produces distinctive highly bunched, truncated fibres, suggesting that the sulphate ions have a sophisticated role to play in fibre formation, rather as they do in the crystal structure. Analytical ultracentrifugation studies show that in the absence of heating, insulin is predominantly dimeric in mineral acids, whereas in acetic acid the equilibrium is shifted towards the monomer. Hence, the effect of acid on the aggregation state of insulin is also complex. These results suggest that acid conditions increase the susceptibility of the molecule to conformational change and dissociation, and enhance the rate of fibrillation by providing a charged environment in which the attractive forces between the protein molecules is increased.     

Mechanism of the fluorescent light induced suppression of Curly phenotype in Drosophila melanogaster

A dominant mutation Curly (Cy), frequently used as a marker on the second chromosome in Drosophila melanogaster, was previously shown to be suppressed by several factors, including larval crowding, low temperature, and fluorescent light. While the first two factors affect this mutation only partially, fluorescent tube exposed flies exhibit an almost completely suppressed (wild type) phenotype. This suppressive effect is the result of a combination of the electric field and light, both factors being produced by common fluorescent tubes. In this study, experiments were carried out to clarify the basic mechanism of this unique phenomenon. Two fluorescent tube sensitive stages of Drosophila development were found in the second half of embryonic development and first half of the pupal stage. Riboflavin, which is administered to Drosophila larvae with yeast, and decomposed by light, seems to play a key role in this phenomenon. In a medium lacking riboflavin caused by light exposure, Cy expression is inhibited by the action of electric field. Positive results of experiments with lithium ions, which block the opening of Ca(2+) channels, support the hypothesis that electromagnetic fields may alter ion currents during ontogenic development of Drosophila, and thus influence, expression of the Cy gene. Also, fluorescent light induces an overexpression of a specific protein in the imaginal wing disc of Cy pupae.     

Effects of 5 s exposures to a 50 microT, 20 Hz magnetic field on skin conductance and ratings of affect and arousal

This study investigated the effect of a weak magnetic field (50 microT, 20 Hz sinusoidal, 5 s duration) on concurrent perceptions of visual stimuli. Subjects were seated between Helmholtz coils and gave post-exposure ratings for the affective content and arousing nature of presented images. They were blind as to the presence or absence of a simultaneously presented field. Skin conductance and arousal ratings did not show significant differences between experimental and control conditions, but the affective content rating did (P = 0.041), with the images viewed under field exposure being rated as having a more positive affect. Such measures might thus be useful as additional indicators of magnetic field detection. A post-hoc analysis of skin conductance profiles showed that 48% of subjects exhibited a lowering of skin conductance during field exposure, 34% exhibited no apparent reaction, and 17% exhibited an increase. Overall ratings given by each of the groups appeared to relate to these physiological profiles.     

Natural very-low-frequency sferics and headache

Very-low-frequency (VLF) atmospherics or sferics are pulse-shaped alternating electric and magnetic fields which originate from atmospheric discharges (lightning). The objective of the study was threefold: (i) to analyse numerous parameters characterizing the sferics activity with regard to their suitability for field studies, (ii) to identify meteorological processes related to the sferics activity and (iii) to investigate the possible association of sferics with pain processes in patients suffering from migraine- and tension-type headaches. Over a period of 6 months (July through December) the sferics activity in the area of Giessen (Germany) was recorded. Three sferics parameters were chosen. The number of sferics impulses per day, the variability of the impulse rate during a day and the variability in comparison to the preceding day were correlated with weather processes (thunderstorm, temperature, vapour pressure, barometric pressure, humidity, wind velocity, warm sector). Significant correlations were obtained during the summer months (July, August) but not during the autumn months (October, November, December). During autumn, however, the sferics activity was correlated with the occurrence of migraine-type headaches (r=0.33, P<0.01) recorded by 37 women who had filled out a headache diary over a period of 6 months (July–December). While the thunderstorm activity was very intense during July and August, no relationship between sferics and migraine was found. In summer, tension-type headaches were associated with meteorological parameters such as temperature (r=0.42, P<0.01) and vapour pressure (r=0.28, P<0.05). Although the sferics activity can explain a small percentage of the variation in migraine occurrence, a direct influence was more likely exerted by visible or otherwise perceptible weather conditions (thunderstorms, humidity, vapour pressure, warm sector, etc.) than by the sferics activity itself. Received: 9 January 2001 / Revised: 1 May 2001 / Accepted: 9 May 2001     

Interaction of estrogen and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in immature male chickens (Gallus domesticus)

The interaction of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and estrogen was studied in chickens to more clearly define this relationship in an avian species and its role in the enhanced sensitivity of female chickens to TCDD-induced wasting syndrome. Twenty male chickens (7-9 weeks old) were divided evenly into four groups: control (CTL, received the same volume of vehicle); estrogen-treated (E2, 1 mg/kg estradiol cypionate injections on days 1, 2 and 3); TCDD-treated (TCDD, single 50 microg/kg injection on day 4); and estrogen plus TCDD (E2+TCDD, as above), with measurements taken on day 14. The E2 group compared with the CTL group had decreased comb height (24%), comb length (26%) and adipose tissue (AT) lipoprotein lipase (LPL) activity relative to AT mass (51%), while liver mass and body weight gain were each increased by 28%. The TCDD group had increased liver mass (62%), reduced comb length (17%), and reduced AT LPL activity indexed to AT mass (70%) compared with the CTL group. Finally, the E2+TCDD group had 37% lower body weight gain and 30% larger livers relative to body mass compared with the E2 group, but were not significantly different from the TCDD group. These data show that TCDD antagonized several effects of exogenous estrogen in male chickens, while estrogen enhanced TCDD toxicity in a tissue-specific manner.     

Spray application technology

Spray application technology on perennial tree crops has been a poorly researched subject. Many of the nozzles and sprayers were originally developed for use on row crops which have an even, flat profile and spraying is downward. Some of the short-comings of these systems were exposed when tree crops were sprayed, particularly the large and dense trees of 30 years ago.Much of the variation experienced in experimental results where fruit trees had been thinned with bioregulators is likely to have been due to application methods. Air-blast sprayers using hydraulic nozzles resulted in inconsistent results causing many research workers to revert to the use of hand lances and small pumps for their trials. Translation of these results to commercial practices then became a problem, as growers were reluctant to revert to the use of hand lances for thinning, particularly as orchards increased in size.The increased emphasis on reducing the use of chemicals, including bioregulators, on food and the need to use ecologically less wasteful methods of applying them has recently promoted more research into spraying techniques.Early work with spray technology identified factors causing variation. Such factors as the range and size of spray droplets, effect of climate, use of wetters, spreaders and stickers, the carrier medium (oil, water or combination), the use of propelled air, the crop species and it's configuration all impacted on the results.     

Proteomic characterization and three-dimensional electron microscopy study of PSII–LHCII supercomplexes from higher plants

In higher plants a variable number of peripheral LHCII trimers can strongly (S), moderately (M) or loosely (L) associate with the dimeric PSII core (C₂) complex via monomeric Lhcb proteins to form PSII–LHCII supercomplexes with different structural organizations. By solubilizing isolated stacked pea thylakoid membranes either with the α or β isomeric forms of the detergent n-dodecyl-D-maltoside, followed by sucrose density ultracentrifugation, we previously showed that PSII–LHCII supercomplexes of types C₂S₂M₂ and C₂S₂, respectively, can be isolated [S. Barera et al., Phil. Trans. R Soc. B 67 (2012) 3389–3399]. Here we analysed their protein composition by applying extensive bottom-up and top-down mass spectrometry on the two forms of the isolated supercomplexes. In this way, we revealed the presence of the antenna proteins Lhcb3 and Lhcb6 and of the extrinsic polypeptides PsbP, PsbQ and PsbR exclusively in the C₂S₂M₂ supercomplex. Other proteins of the PSII core complex, common to the C₂S₂M₂ and C₂S₂ supercomplexes, including the low molecular mass subunits, were also detected and characterized. To complement the proteomic study with structural information, we performed negative stain transmission electron microscopy and single particle analysis on the PSII–LHCII supercomplexes isolated from pea thylakoid membranes solubilized with n-dodecyl-α-D-maltoside. We observed the C₂S₂M₂ supercomplex in its intact form as the largest PSII complex in our preparations. Its dataset was further analysed in silico, together with that of the second largest identified sub-population, corresponding to its C₂S₂ subcomplex. In this way, we calculated 3D electron density maps for the C₂S₂M₂ and C₂S₂ supercomplexes, approaching respectively 30 and 28 Å resolution, extended by molecular modelling towards the atomic level. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Inhibition of the central melanocortin system decreases brown adipose tissue activity

The melanocortin system is an important regulator of energy balance, and melanocortin 4 receptor (MC4R) deficiency is the most common monogenic cause of obesity. We investigated whether the relationship between melanocortin system activity and energy expenditure (EE) is mediated by brown adipose tissue (BAT) activity. Therefore, female APOE*3-Leiden.CETP transgenic mice were fed a Western-type diet for 4 weeks and infused intracerebroventricularly with the melanocortin 3/4 receptor (MC3/4R) antagonist SHU9119 or vehicle for 2 weeks. SHU9119 increased food intake (+30%) and body fat (+50%) and decreased EE by reduction in fat oxidation (−42%). In addition, SHU9119 impaired the uptake of VLDL-TG by BAT. In line with this, SHU9119 decreased uncoupling protein-1 levels in BAT (−60%) and induced large intracellular lipid droplets, indicative of severely disturbed BAT activity. Finally, SHU9119-treated mice pair-fed to the vehicle-treated group still exhibited these effects, indicating that MC4R inhibition impairs BAT activity independent of food intake. These effects were not specific to the APOE*3-Leiden.CETP background as SHU9119 also inhibited BAT activity in wild-type mice. We conclude that inhibition of central MC3/4R signaling impairs BAT function, which is accompanied by reduced EE, thereby promoting adiposity. We anticipate that activation of MC4R is a promising strategy to combat obesity by increasing BAT activity.