Effect of streptavidins with varying biotin binding affinities on the properties of biotinylated gramicidin channels

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.     

Synergistic effect of hydrogen peroxide and elastase on elastic fiber injury in vitro

This laboratory has previously shown that hyperoxia enhances airspace enlargement in a hamster model of elastase-induced emphysema. To further understand the mechanism responsible for this finding, the effect of oxidants on elastase activity was studied in vitro, using a radiolabeled elastic fiber matrix derived from rat pleural mesothelial cells. Matrix samples were treated with either 0.1%, 1%, 3%, or 10% hydrogen peroxide (H2O2) for 1 hr, then incubated with 1.0 microg/ml porcine pancreatic elastase for 2 hrs. Radioactivity released from the matrix was used as a measure of elastolysis. Results indicate that sequential exposure to H2O2 and elastase markedly enhanced elastolysis compared to enzyme treatment alone. A 22% increase in elastolysis was seen with 0.1% H2O2 (325 vs. 396 cpm; P < 0.05), whereas samples pretreated with 1%, 3%, and 10% H2O2 showed increases of 53% (274 vs. 420 cpm; P < 0.05), 71% (381 vs. 653 cpm; P < 0.01), and 38% (322 vs. 443 cpm; P < 0.01), respectively. Exposure to various concentrations of H2O2 alone (0.1% to 10%) produced only minimal elastolysis (<20 cpm). However, 1% H2O2 was capable of degrading peptide-free desmosine and isodesmosine, suggesting that exposure to this oxidant may reduce the stability of the elastic fiber matrix. With regard to lung diseases such as emphysema, H2O2 and other oxidants derived from inflammatory cells or the environment could possibly act as priming agents for elastase-mediated breakdown of elastic fibers, resulting in amplification of lung injury.     

Interaction of fraternal birth order and handedness in the development of male homosexuality

The present study investigated evidence for an interaction between two of the best established etiologic factors, or markers of etiologic factors, in the literature on male homosexuality: fraternal birth order and hand preference. By combining five samples, the authors produced study groups of 1774 right-handed heterosexuals, 287 non-right-handed heterosexuals, 928 right-handed homosexuals, and 157 non-right-handed homosexuals. The results showed a significant (P = 0.004) handedness by older brothers interaction, such that (a) the typical positive correlation between homosexuality and greater numbers of older brothers holds only for right-handed males, (b) among men with no older brothers, homosexuals are more likely to be non-right-handed than heterosexuals; among men with one or more older brothers, homosexuals are less likely to be non-right-handed than heterosexuals, and (c) the odds of homosexuality are higher for men who have a non-right hand preference or who have older brothers, relative to men with neither of these features, but the odds for men with both features are similar to the odds for men with neither. These findings have at least two possible explanations: (a) the etiologic factors associated with non-right-handedness and older brothers-hypothesized to be hyperandrogenization and anti-male antibodies, respectively-counteract each other, yielding the functional equivalent of typical masculinization, and (b) the number of non-right-handed homosexuals with older brothers is smaller than expected because the combination of the older brothers factor with the non-right-handedness factor is toxic enough to lower the probability that the affected fetus will survive.     

Small amounts of isotope-reinforced polyunsaturated fatty acids suppress lipid autoxidation

Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D(2)-Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast.     

Biotic versus abiotic pollination in Oeceoclades maculata (Lindl.) Lindl. (Orchidaceae)

Geographical variation in the reproductive biology of widespread species often occurs at their distributional boundaries. We sought to determine whether such variation has occurred in an invasive orchid, Oeceoclades maculata, across its naturalized range. We compared its reproductive biology in a Brazilian population with that published for a population on the Caribbean island of Puerto Rico. In the state of São Paulo, O. maculata flowers between December and February, at the height of the rainy season. Similar fruit sets were observed in manual self (76%) and cross (70.4%) pollination treatments. The fruit set of plants protected from both pollinators and rainfall was 6.1%, whereas plants exposed only to rainfall had a fruit set of 41.4%, slightly less than the controls (48.3%). Like the Puerto Rico population, reproduction is primarily through rain‐assisted autogamy, but unlike observations made on the island, outcrossing can eventually occur. We observed two butterfly species (Heliconius ethilla narcaea and Heliconius erato phyllis) pollinating O. maculata. Secretory epidermal cells and trichomes of the spur lumen produced 0.7 µL of 25% (sucrose equivalents) nectar per flower each morning, which was stored in a dilated basal portion of the spur and reabsorbed by the afternoon. Thus, geographical variation in reproductive biology exists across the broad invasive range of O. maculata.     

Effect of light stress from phytoplankton on the relationship between aquatic vegetation and the propagule bank in shallow lakes

1. The way light stress controls the recruitment of aquatic plants (phanerogams and charophytes) is a key process controlling plant biodiversity, although still poorly understood. Our aim was to investigate how light stress induced by phytoplankton, that is, independent from the aquatic plants themselves, determines the recruitment and establishment of plant species from the propagule bank. The hypotheses were that an increase in light stress (i) decreases abundance and species richness both of established aquatic plants and of propagules in the bank and (ii) decreases the recruitment success of plants from this bank. 2. These hypotheses were tested in 25 shallow lakes representing a light stress gradient, by sampling propagule banks before the recruitment phase and when the lakes are devoid of actively growing plants (i.e. at the end of winter), established vegetation at the beginning of the summer and phytoplankton biomass (chlorophyll a) during the recruitment and establishment phase. 3. The phytoplankton biomass was negatively correlated with the richness and abundance of established vegetation but was not correlated with the propagule bank (neither species richness nor propagule abundance). The similarity between the propagule bank and established vegetation decreased significantly with increasing phytoplankton biomass. 4. The contrast in species composition between the vegetation and the propagule bank at the highest light stress suggests poor recruitment from the propagule bank but prompts questions about its origin. It could result from dispersal of propagules from neighbouring systems. Propagules could also originate from a persistent propagule bank formerly produced in the lake, suggesting strong year‐to‐year variation in light stress and, as a consequence, in recruitment and reproductive success of plants.