Membrane assembly of M13 major coat protein: evidence for a structural adaptation in the hinge region and a tilted transmembrane domain

New insights into the low-resolution structure of the hinge region and the transmembrane domain of the membrane-bound major coat protein of the bacteriophage M13 are deduced from a single cysteine-scanning approach using fluorescence spectroscopy. New mutant coat proteins are labeled and reconstituted into phospholipid bilayers with varying headgroup compositions (PC, PE, and PG) and thicknesses (14:1PC, 18:1PC, and 22:1PC). Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. It is found that the protein is almost entirely embedded in the membrane, whereas the phospholipid headgroup composition of the membrane hardly affects the overall embedment of the protein in the membrane. From the assessment of a hydrophobic and hydrophilic face of the transmembrane helix, it is concluded that the helix is tilted with respect to the membrane normal. As compared to the thicker 18:1PC and 22:1PC membranes, reconstitution of the protein in the thin 14:1PC membranes results in a loss of helical structure and in the formation of a stretched conformation of the hinge region. It is suggested that the hinge region acts as a flexible spring between the N-terminal amphipathic arm and transmembrane hydrophobic helix. On average, the membrane-bound state of the coat protein can be seen as a gently curved and tilted, "banana-shaped" molecule, which is strongly anchored in the membrane-water interface at the C-terminus. From our experiments, we propose a rather small conformational adaptation of the major coat protein as the most likely reversible mechanism for responding to environmental changes during the bacteriophage disassembly and assembly process.     

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger.

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pH(cyt)) and vacuolar pH (pH(vac)) values of 7.6 and 6.2, respectively, when the extracellular pH (pH(ex)) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a Delta pH over the cytoplasmic membrane of 6.6-6.7 was reached (pH(ex) 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH(ex) 5.8. Perfusion in the presence of various hexoses and pentoses (pH(ex) 5.8) revealed that the magnitude of Delta pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger Delta pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 microm CCCP), transient (2 microm CCCP) or permanent (10 microm CCCP) collapse of the vacuolar membrane Delta pH. Nonlethal levels of the metabolic inhibitor azide (N3-, 0.1 mm) caused a transient decrease in pH(cyt) that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH(ex) (1.8) and a high acid-secreting capacity, pH(cyt) and pH(vac) values were still well maintained (pH 7.5 and 6.4, respectively).     

MAIT Cells Detect and Efficiently Lyse Bacterially-Infected Epithelial Cells

Mucosal associated invariant T cells (MAIT) are innate T lymphocytes that detect a large variety of bacteria and yeasts. This recognition depends on the detection of microbial compounds presented by the evolutionarily conserved major-histocompatibility-complex (MHC) class I molecule, MR1. Here we show that MAIT cells display cytotoxic activity towards MR1 overexpressing non-hematopoietic cells cocultured with bacteria. The NK receptor, CD161, highly expressed by MAIT cells, modulated the cytokine but not the cytotoxic response triggered by bacteria infected cells. MAIT cells are also activated by and kill epithelial cells expressing endogenous levels of MRI after infection with the invasive bacteria Shigella flexneri. In contrast, MAIT cells were not activated by epithelial cells infected by Salmonella enterica Typhimurium. Finally, MAIT cells are activated in human volunteers receiving an attenuated strain of Shigella dysenteriae-1 tested as a potential vaccine. Thus, in humans, MAIT cells are the most abundant T cell subset able to detect and kill bacteria infected cells.     

Structural and magnetic phase transitions in simple oxides using hybrid functionals

We present the structural as well as elastic properties of the alkaline earth oxides and FeO, calculated using hybrid exchange functionals within DFT. We show that by empirically fitting the amount of Fock-exchange in the hybrid functionals, we can accurately reproduce the pressure-induced phase transitions for MgO, CaO, SrO and BaO. For FeO the hybrid functionals predict an insulator?metal transition at ca. 150?GPa, associated with an i-B8?B8 structural phase transition. The structural phase transition is accompanied by a spin transition from a high- to low-spin electron configuration on the Fe²⁺ ions. Hence, FeO undergoes a magnetic phase transition from an anti-ferromagnetic to non-magnetic structure. We also find that as the ionicity of the polymorphs increases a higher fraction of Fock-exchange is required to reproduce the structural volumes reported from experiments.     

Predicting demographically sustainable rates of adaptation: can great tit breeding time keep pace with climate change?

Populations need to adapt to sustained climate change, which requires micro-evolutionary change in the long term. A key question is how the rate of this micro-evolutionary change compares with the rate of environmental change, given that theoretically there is a ‘critical rate of environmental change’ beyond which increased maladaptation leads to population extinction. Here, we parametrize two closely related models to predict this critical rate using data from a long-term study of great tits (Parus major). We used stochastic dynamic programming to predict changes in optimal breeding time under three different climate scenarios. Using these results we parametrized two theoretical models to predict critical rates. Results from both models agreed qualitatively in that even ‘mild’ rates of climate change would be close to these critical rates with respect to great tit breeding time, while for scenarios close to the upper limit of IPCC climate projections the calculated critical rates would be clearly exceeded with possible consequences for population persistence. We therefore tentatively conclude that micro-evolution, together with plasticity, would rescue only the population from mild rates of climate change, although the models make many simplifying assumptions that remain to be tested.     

Copolymers from unsaturated macrolactones: toward the design of cross-linked biodegradable polyesters

The enzymatic synthesis of a series of random copolyesters by ring-opening polymerization of unsaturated macrolactones like globalide and ambrettolide with 1,5-dioxepan-2-one (DXO) and 4-methyl caprolactone (4MeCL) was investigated. (13)C NMR diad analysis confirmed the randomness of all copolymers irrespective of the comonomer ratios. Thermal investigation showed that incorporating the comonomers lowered the melting points of the polymers as compared with the macrolactone homopolymers. The decrease was dependent on the comonomer ratio. The unsaturated copolymers were thermally cross-linked using dicumyl peroxide, which resulted in completely amorphous insoluble networks. It was found that 10% incorporation of the unsaturated macolactone was sufficient to obtain a gel content of 95 wt %. Preliminary degradation tests confirm that the cross-linked copolymers are enzymatically degradable and that the incorporation of hydrophilic comonomers like DXO enhances degradation.     

Assumed White Blood Cell Count of 8,000 Cells/μL Overestimates Malaria Parasite Density in the Brazilian Amazon

Quantification of parasite density is an important component in the diagnosis of malaria infection. The accuracy of this estimation varies according to the method used. The aim of this study was to assess the agreement between the parasite density values obtained with the assumed value of 8,000 cells/μL and the automated WBC count. Moreover, the same comparative analysis was carried out for other assumed values of WBCs. The study was carried out in Brazil with 403 malaria patients who were infected in different endemic areas of the Brazilian Amazon. The use of a fixed WBC count of 8,000 cells/μL to quantify parasite density in malaria patients led to overestimated parasitemia and resulted in low reliability when compared to the automated WBC count. Assumed values ranging between 5,000 and 6,000 cells/μL, and 5,500 cells/μL in particular, showed higher reliability and more similar values of parasite density when compared between the 2 methods. The findings show that assumed WBC count of 5,500 cells/μL could lead to a more accurate estimation of parasite density for malaria patients in this endemic region.     

Effects of Work and Orcadian Rhythm on Bus Drivers' Oral Temperature

The results described in this paper originate from a research project aiming at the development of a useful measuring instrument which can demonstrate the effect of work and work circumstances on the task performer. Among other physiological (and psychometric) variables, oral temperature data were obtained from eight younger and eight older bus drivers working in various shifts. Measurements were performed in an experimental design under standardized conditions in a mobile laboratory before starting, during some rest intervals, and after finishing work. The same measurements were performed on the bus drivers in a control condition at corresponding times on a day off. The results indicated that only during the working days a temperature pattern could be detected resembling the data found in the literature (low in the morning, a maximum in the late afternoon, then a decline). No clear pattern was discernible on work-free days. On working days the mean oral temperature values were significantly higher at corresponding hours of the day. Upward deviations of the temperature pattern were found before starting work (but also at the beginning of the day off), while lower values were obtained after finishing work (especially in the shifts starting in the morning). These results might be interpreted in terms of interaction between circadian rhythm and activating and de-activating tendencies connected with (structured) daily activities.     

Effects of 17-β-estradiol treatment of female zebra finches on offspring sex ratio and survival

Treatment of female zebra finches (Taeniopygia guttata) with 17-beta-estradiol leads to a female-biased sex ratio in their offspring at the age of independence [Horm. Behav. 35 (1999) 135]. It is unclear whether this is due to a bias of the primary sex ratio or to sex-specific survival. We replicated this experiment and found again a significantly higher total number of daughters than sons at independence in the estradiol-treated group. This was due to higher embryonic survival of daughters compared with sons in the estradiol-treated group and the reverse in the control group. There was no effect of the hormone treatment on the primary sex ratio. Treatment with 17-beta-estradiol led to a significantly shorter hatching time and to heavier offspring at day 7 after hatching. This weight was correlated with maternal plasma estradiol levels on the day of the first egg, which were significantly higher in the estradiol-treated group than in the control group. The results do not support the idea that maternal estradiol levels influence the primary sex ratio. They indicate that maternal estradiol differentially affects survival of sons and daughters via an influence on the embryonic environment, possibly enhancing offspring growth.