Elastic deformations of the rotary double motor of single F(o)F(1)-ATP synthases detected in real time by Förster resonance energy transfer

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, F?rster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to a 3-stepped F(1) motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F(O)F(1)-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Looking for a similar partner: host plants shape mating preferences of herbivorous insects by altering their contact pheromones

The role of phenotypical plasticity in ecological speciation and the evolution of sexual isolation remains largely unknown. We investigated whether or not divergent host plant use in an herbivorous insect causes assortative mating by phenotypically altering traits involved in mate recognition. We found that males of the mustard leaf beetle Phaedon cochleariae preferred to mate with females that were reared on the same plant species to females provided with a different plant species, based on divergent cuticular hydrocarbon profiles that serve as contact pheromones. The cuticular hydrocarbon phenotypes of the beetles were host plant specific and changed within 2 weeks after a shift to a novel host plant species. We suggest that plant-induced phenotypic divergence in mate recognition cues may act as an early barrier to gene flow between herbivorous insect populations on alternative host species, preceding genetic divergence and thus, promoting ecological speciation.     

Refractive Index Sensor Using Long-Range Surface Plasmon Resonance with Prism Coupler

Plasmonics - Long-range surface plasmon resonance (LRSPR)-based sensors exhibit high sensitivity as compared to the conventional SPR sensors due to low losses. A high refractive index prism and low...     

Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains

For optimization of the transformation procedure with Pisum sativum L. stern explant callus was used to test the effect of disarmed Agrobacterium tumefaciens strains, cocultivation procedures (preconditioning of explants; use of Nicotiana tabacum L. nurse cultures), duration of cocultivation (2, 3 or 4 days), and agents for selection (kanamycin or hygromycin). The succinamopine strain EHA101(pBI1042) produced the highest percentage of transformed calli (77%) when used in conjunction with tobacco nurse culture during four days of cocultivation. Using this strain, kanamycin (76%) and hygromycin (77%) were equally effective selective agents, but for strain LBA4404(pBI1042) percentage of transformed calli was higher for hygromycin (63%) than for kanamycin (17%). The procedures and strains shown to be optimal for transformation of pea callus will now be complemented by a pea regeneration system.     

Phenology of first and peak emergence of Aphthona lacertosa and A. nigriscutis: Two flea beetles introduced for biological control of leafy spurge, Euphorbia esula L.

Nonlinear models were used to estimate first emergence and peak abundance dates for Aphthona lacertosa Rosenhauer and A. nigriscutis Foudras, two flea beetles introduced to control leafy spurge, Euphorbia esula L., in North America. For model development, 26 field sites were sampled for flea beetle abundance at weekly intervals for eight weeks in three western Minnesota counties in 2000, 2001, and 2002. A three-parameter Weibull function, fit to observed cumulative probability distributions, were used to predict accumulated degree-days (ADD) to first emergence. Bias testing indicated the Weibull function provided a useful estimate of first emergence for A. lacertosa (304 ADD, lower developmental threshold 7.5 °C), but failed to produce a useful estimate for A. nigriscutis. A third-order polynomial was used to approximate seasonal abundance and predict peak abundance for each species. Estimated ADD to peak abundance of A. lacertosa was 594 ± 24 (DD > 7.5 °C) and 670 ± 15 (DD > 9.3 °C) for A. nigriscutis. Models were validated with additional data sets from Minnesota, Montana, and North Dakota. Estimated date of peak emergence provided useful predictions of peak emergence for Minnesota and North Dakota, but failed to predict peak emergence in Montana. We speculate that variation in climate and environmental conditions between Midwestern states and Montana were responsible for differing emergence patterns. We conclude that phenology models should be developed regionally to provide useful predictions of peak emergence for land managers. Maps were developed for Minnesota to spatially display predicted dates of peak abundance for A. lacertosa and A. nigriscutis.     

Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.     

Effects of six years atmospheric CO2 enrichment on plant,soil, and soil microbial C of a calcareous grassland

Stimulated plant production and often even larger stimulation of photosynthesis at elevated CO₂ raise the possibility of increased C storage in plants and soils. We analysed ecosystem C partitioning and soil C fluxes in calcareous grassland exposed to elevated CO₂ for 6 years. At elevated CO₂, C pools increased in plants (+23%) and surface litter (+24%), but were not altered in microbes and soil organic matter. Soils were fractionated into particle size and density separates. The amount of low-density macroorganic C, an indicator of particulate soil C inputs from root litter, was not affected by elevated CO₂. Incorporation of C fixed during the experiment (C_new) was tracked by C isotopic analysis of soil fractions which were labelled due to ¹³C depletion of the commercial CO₂ used for atmospheric enrichment. This data constrains estimates of C sequestration (absolute upper bound) and indicates where in soils potentially sequestered C is stored. C_new entered soils at an initial rate of 210±42 g C m^–2 year^–1, but only 554±39 g C_new m^–2 were recovered after 6 years due to the low mean residence time of 1.8 years. Previous process-oriented measurements did not indicate increased plant–soil C fluxes at elevated CO₂ in the same system (¹³C kinetics in soil microbes and fine roots after pulse labelling, and minirhizotron observations). Overall experimental evidence suggests that C storage under elevated CO₂ occurred only in rapidly turned-over fractions such as plants and detritus, and that potential extra soil C inputs were rapidly re-mineralised. We argue that this inference does not conflict with the observed increases in photosynthetic fixation at elevated CO₂, because these are not good predictors of plant growth and soil C fluxes for allometric reasons. C sequestration in this natural system may also be lower than suggested by plant biomass responses to elevated CO₂ because C storage may be limited by stabilisation of C_new in slowly turned-over soil fractions (a prerequisite for long-term storage) rather than by the magnitude of C inputs per se.     

Mexiletine and its Interactions with Classical Antiepileptic Drugs: An Isobolographic Analysis

Using the mouse maximal electroshock test, the reference model of tonic–clonic seizures, the aim of the present study was to determine the type of interaction between mexiletine (a class IB antiarrhythmic drug) and classical antiepileptics: valproate, carbamazepine, phenytoin, and phenobarbital. Isobolographic analysis of obtained data indicated antagonistic interactions between mexiletine and valproate (for fixed ratio combinations of 1:1 and 3:1). Additivity was observed between mexiletine and valproate applied in proportion of 1:3 as well as between mexiletine and remaining antiepileptics for the fixed ratios of 1:3, 1:1, and 3:1. Neither motor performance nor long-term memory were impaired by mexiletine or antiepileptic drugs regardless of whether they were administered singly or in combination. Mexiletine did not significantly affected brain concentrations of carbamazepine, phenobarbital or phenytoin. In contrast, the antiarrhythmic drug decreased by 23 % the brain level of valproate. This could be, at least partially, the reason of antagonistic interaction between the two drugs. In conclusion, the observed additivity suggests that mexiletine can be safely applied in epileptic patients treated with carbamazepine, phenytoin or phenobarbital. Because of undesirable pharmacodynamics and pharmacokinetic interactions with valproate, mexiletine should not be used in such combinations.

     

The Salivary Scavenger and Agglutinin (SALSA) in Healthy and Complicated Pregnancy

Pre-eclampsia is a leading cause of maternal and perinatal morbidity and mortality worldwide. The etiology is not clear, but an immune attack towards components of placenta or fetus has been indicated. This involves activation of the complement system in the placenta. We have previously described the presence of the complement-regulating protein salivary scavenger and agglutinin (SALSA) in amniotic fluid. In this study we investigated the potential role of SALSA in pregnancy by analyzing its presence in amniotic fluid and placental tissue during healthy and complicated pregnancies. SALSA levels in amniotic fluid increased during pregnancy. Before 20 weeks of gestation the levels were slightly higher in patients who later developed pre-eclampsia than in gestation age-matched controls. In the placenta of pre-eclamptic patients syncytial damage is often followed by the formation of fibrinoid structures. SALSA was found clustered into these fibrinoid structures in partial co-localization with complement C1q and fibronectin. In vitro analysis showed direct protein binding of SALSA to fibronectin. SALSA binds also to fibrin/fibrinogen but did not interfere with the blood clotting process in vitro. Thus, in addition to antimicrobial defense and epithelial differentiation, the data presented here suggest that SALSA, together with fibronectin and C1q, may be involved in the containment of injured placental structures into fibrinoids.     

Incidence and Risk Factors for Respiratory Syncytial Virus and Human Metapneumovirus Infections among Children in the Remote Highlands of Peru

Introduction

The disease burden and risk factors for respiratory syncytial virus (RSV) and human metapneumovirus (MPV) infections among children living in remote, rural areas remain unclear.

Materials and Methods

We conducted a prospective, household-based cohort study of children aged <3 years living in remote rural highland communities in San Marcos, Cajamarca, Peru. Acute respiratory illnesses (ARI), including lower respiratory tract infection (LRTI), were monitored through weekly household visits from March 2009 through September 2011. Nasal swabs collected during ARI/LRTI were tested for RSV, MPV, and other respiratory viruses using real-time RT-PCR. Incidence rates and rate ratios were calculated using mixed effects Poisson regression.

Results

Among 892 enrolled children, incidence rates of RSV and MPV ARI were 30 and 17 episodes per 100 child-years, respectively. The proportions of RSV and MPV ARI that presented as LRTI were 12.5% and 8.9%, respectively. Clinic visits for ARI and hospitalizations were significantly more frequent (all p values <0.05) among children with RSV (clinic 41% and hospital 5.3%) and MPV ARI (38% and 3.5%) when compared with other viral infections (23% and 0.7%) and infections without virus detected (24% and 0.6%). In multivariable analysis, risk factors for RSV detection included younger age (RR 1.02, 95% CI: 1.00-1.03), the presence of a smoker in the house (RR 1.63, 95% CI: 1.12-2.38), residing at higher altitudes (RR 1.93, 95% CI: 1.25-3.00 for 2^nd compared to 1^st quartile residents; RR 1.98, 95% CI: 1.26-3.13 for 3^rd compared to 1^st quartile residents). Having an unemployed household head was significantly associated with MPV risk (RR 2.11, 95% CI: 1.12-4.01).

Conclusion

In rural high altitude communities in Peru, childhood ARI due to RSV or MPV were common and associated with higher morbidity than ARI due to other viruses or with no viral detections. The risk factors identified in this study may be considered for interventional studies to control infections by these viruses among young children from developing countries.