DNA uptake in swine sperm: Effect of plasmid topology and methyl‐beta‐cyclodextrin‐mediated cholesterol depletion

Sperm‐mediated gene transfer (SMGT), the ability of sperm cells to spontaneously incorporate exogenous DNA and to deliver it to oocytes during fertilization, has been proposed as an easy and efficient method for producing transgenic animals. SMGT is still undergoing development and optimization to improve the uptake efficiency of foreign DNA by sperm cells, which is a preliminary, yet critical, step for successful SMGT. Towards this aim, we developed a quantitative, real‐time PCR‐based assay to assess the absolute number of exogenous plasmids internalized into the spermatozoon. Using this technique, we found that the circular form of the DNA is more efficiently taken up than the linearized form. We also found that DNA internalization into the nucleus of porcine sperm cells is better under specific methyl‐β‐cyclodextrin (MCD)‐treated conditions, where the plasma membrane properties were altered without significantly compromising sperm physiology. These results provide the first evidence that membrane cholesterol depletion by MCD might represent a novel strategy for enhancing the ability of sperm to take up heterologous DNA. Mol. Reprod. Dev. 79: 853–860, 2012. © 2012 Wiley Periodicals, Inc.     

Influence of positive allosteric modulators on GABA(B) receptor coupling in rat brain: a scintillation proximity assay characterisation of G protein subtypes

Little is known concerning coupling of cerebral GABA_B receptors to G protein subtypes, and the influence of positive allosteric modulators (PAMs) has not been evaluated. These questions were addressed by an antibody-capture/scintillation proximity assay strategy. GABA concentration-dependently enhanced the magnitude of [³⁵S]GTPγS binding to Gαo and, less markedly, Gαi_1/3 in cortex, whereas Gq and Gs/olf were unaffected. ( R )-baclofen and SKF97581 likewise activated Gαo and Gαi_1/3, expressing their actions more potently than GABA. Similar findings were acquired in hippocampus and cerebellum, and the GABA_B antagonist, CGP55845A, abolished agonist-induced activation of Gαo and Gαi_1/3 in all structures. The PAMs, GS39783, CGP7930 and CGP13501, inactive alone, enhanced efficacy and potency of agonist-induced [³⁵S]GTPγS binding to Gαo in all regions, actions abolished by CGP55845A. In contrast, they did not modify efficacies at Gαi_1/3. Similarly, in human embryonic kidney cells expressing GABA_B(1a+2) or GABA_B(1b+2) receptors, allosteric modulators did not detectably enhance efficacy of GABA at Gαi_1/3, though they increased its potency. To summarise, GABA_B receptors coupled both to Gαo and to Gαi, but not Gq and Gs/olf, in rat brain. PAMs more markedly enhanced efficacy of coupling to Go versus Gi_1/3. It will be of interest to confirm these observations employing complementary techniques and to evaluate their potential therapeutic significance.     

Regulatory signals for endothelial podosome formation

Podosomes are punctate actin-rich adhesion structures which spontaneously form in cells of the myelomonocytic lineage. Their formation is dependent on Src and RhoGTPases. Recently, podosomes have also been described in vascular cells. These podosomes differ from the former by the fact that they are inducible. In endothelial cells, such a signal can be provided by either constitutively active Cdc42, the PKC activator PMA or TGFbeta, depending on the model. Consequently, other regulatory pathways have been reported to contribute to podosome formation. To get more insight into the mechanisms by which podosomes form in endothelial cells, we have explored the respective contribution of signal transducers such as Cdc42-related GTPases, Smads and PKCs in three endothelial cell models. Results presented demonstrate that, in addition to Cdc42, TC10 and TCL GTPases can also promote podosome formation in endothelial cells. We also show that PKCalpha can be either necessary or entirely dispensable, depending on the cell model. In contrast, PKCdelta is essential for podosome formation in endothelial cells but not smooth muscle cells. Finally, although podosomes vary very little in their molecular composition, the signalling pathways involved in their assembly appear very diverse.     

Structural properties of plant and mammalian lipoxygenases. Temperature-dependent conformational alterations and membrane binding ability

Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases with major health and political relevance (atherosclerosis, osteoporosis). The crystal structures of various lipoxygenase-isoforms have been reported, and X-ray coordinates for enzyme-ligand complexes are also available. Although the 3D-structures of plant and animal lipoxygenase-isoforms are very similar, recent small-angle X-ray scattering data suggested a higher degree of motional flexibility of mammalian isozymes in aqueous solutions. To explore the molecular basis for these differences we performed dynamic fluorescence measurements that allowed us to study temperature-induced conformational changes arising from three-dimensional fluctuations of the protein matrix. For this purpose, we first investigated the impact of elevated temperature on activity, secondary structure, tertiary structure dynamics and conformational alterations. Applying fluorescence resonance energy transfer we also tested the membrane binding properties of the two lipoxygenase-isoforms, and compared their binding parameters. Taken together, our results indicate that the rabbit 12/15-lipoxygenase is more susceptible to temperature-induced structural alterations than the soybean enzyme. Moreover, the rabbit enzyme exhibits a higher degree of conformational flexibility of the entire protein molecule (global flexibility) and offers the possibility of augmented substrate movement at the catalytic center (local flexibility).     

How does pollination mutualism affect the evolution of prior self‐fertilization? A model

The mode of pollination is often neglected regarding the evolution of selfing. Yet the distribution of mating systems seems to depend on the mode of pollination, and pollinators are likely to interfere with selfing evolution, since they can cause strong selective pressures on floral traits. Most selfing species reduce their investment in reproduction, and display smaller flowers, with less nectar and scents (referred to as selfing syndrome). We model the evolution of prior selfing when it affects both the demography of plants and pollinators and the investment of plants in pollination. Including the selfing syndrome in the model allows to predict several outcomes: plants can evolve either toward complete outcrossing, complete selfing, or to a stable mixed‐mating system, even when inbreeding depression is high. We predict that the evolution to high prior selfing could lead to evolutionary suicides, highlighting the importance of merging demography and evolution in models. The consequence of the selfing syndrome on plant–pollinator interactions could be a widespread mechanism driving the evolution of selfing in animal‐pollinated taxa.     

Egg trading in the simultaneously hermaphroditic polychaete worm Ophryotrocha gracilis (Huth)

Ophryotrocha gracilis (Huth 1934) is a simultaneously hermaphroditicpolychaete worm with external fertilization and a brief protandrousphase. The mating system of this species seems to meet conditionsleading to the establishment of egg-trading behavior. Experimentsshowed that mating occurs in pairs composed of two simultaneoushermaphrodites; sex roles are sequentially alternated and self-fertilizationis avoided. Egg reciprocation is kept evolutionarily stableby laying eggs in multiple, small-sized egg clutches and reducingthe reproductive success of pairs in which one of the two partnersdoes not reciprocate egg exchange. The frequency of ovigeroushermaphrodites in mass cultures of O. gracilis is about 50".Such a high mate availability preadapts hermaphrodites of O.gracilis to change partners very frequently and to reduce investmentin parental care, contrary to what is observed in another egg-trading,simultaneously hermaphroditic species, O. diadema. Laboratorypopulations of O. diadema have a frequency of only 17% ovigeroushermaphrodites     

Reduced sexual dichromatism,mutual ornamentation,and individual quality in the monogamous Zenaida dove Zenaida aurita

Although variation in plumage coloration is known to occur both between and within sexes, its study remains limited to a few bird families. The Zenaida dove Zenaida aurita is a socially monogamous tropical columbid bird species, characterized by an overall cinnamon‐brownish plumage and structural colorations on the head and neck. The species has been described as sexually dichromatic for plumage, although color differences between males and females are not obvious in the field. We investigated variation in the presumably melanin‐based color of the crown, mantle, breast, and belly, in the iridescent dark‐blue streaks on the head, and in the symmetric iridescent patches on the neck, over the whole spectrum visible to birds. Further, unlike most previous studies, we assessed covariation between plumage color and phenotypic traits in both males and females in relation to the putative signaling function of ornaments. Zenaida doves appeared to be slightly sexually dichromatic for the hue of pigment‐based colored areas, with males being on average more reddish than females. However, this difference was not discernible when considering the avian visual system. Conversely, although the reflectance spectra of iridescent plumage did not significantly differ between sexes in brightness, chroma or spectral position of the peaks, color discrimination analyses showed that individuals should be able to perceive between‐ or within‐sex differences in the color of the iridescent patch. In addition, several color parameters of brown and iridescent feathers were significantly related to territorial status, body condition, wing chord, and, albeit weakly, to individual multilocus heterozygosity. Overall, our results thus suggest that plumage color might be a reliable signal of quality in individuals of both sexes in this species. Further studies are needed to test the potential implication of plumage coloration in mate choice and mating patterns in the Zenaida dove.     

Changes in Hg fractionation in soil induced by willow

This study investigated the effect of willow (Salix viminalis ×  S. schwerinii) on soil characteristics, including changes in Hg fractionation in the soil solid phase, and Hg accumulation and distribution in pot-grown plants cultivated for 32 and 76 days in aged Hg-contaminated soil (30 mg Hg kg⁻¹DW). Changes in soil pH and organic carbon content as well as in Hg fractionation were monitored in both rhizospheric soil and in soil without plants. Mercury fractionation was performed by a 5-step sequential soil extraction procedure. Organic carbon content increased while pH decreased in the rhizospheric soil. Both chemically defined exchangeable Hg (0.1) and Hg bound to humic and fulvic acids (1.1) decreased in the rhizospheric soil, whereas plant accumulation of Hg increased with cultivation time. The sum of the decrease of these two soil Hg fractions after 76 days of cultivation was approximately equal to the amount of Hg accumulated in plants. Furthermore, the major Hg fractions (Hg bound to residual organic matter (53), sulphides (43), and the residual fraction (2.5)) remained stable. Neither whole plant accumulation of Hg from the soil, approximately 0.2 of total Hg in soil after 76 days cultivation, nor the fraction of total plant Hg in the shoots, which accounted for about 3 of the total plant Hg pool regardless of the cultivation time, were high. The overall results suggest that plants might be suitable for phytostabilization of aged Hg-contaminated soil, where root systems trap bioavailable Hg and help to control both leaching of Hg and re-entrainment of Hg-containing particulates from a contaminated site.     

Modulations of the amide function of the preferential dopamine D3 agonist (R,R)-S32504: Improvements of affinity and selectivity for D3 versus D2 receptors

Starting with the preferential dopamine (DA) D₃ agonist S32504, we prepared two series of derivatives of the general formula I-A and I-B, in an effort to improve both potency and selectivity. For the first set of derivatives, where the primary amide function of S32504 was replaced by either secondary and tertiary amide or ester, acid, nitrile and ketone, no improvement was obtained. Conversely, when the primary amide function was integrated in a lactam ring, an enhancement of affinity and selectivity was attained for the five-membered ring lactam but also for its five-membered ring lactone analogue.