A modular design for the clathrin- and actin-mediated endocytosis machinery

Endocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.     

Occurrence of the pugnose pipefish Bryx dunckeri in the Sargasso Sea

Juvenile specimens of the pugnose pipefish, Bryx dunckeri, were collected during a multipurpose research survey conducted within the Sargasso Sea Subtropical Convergence Zone, extending the known distribution range of this species to include open ocean areas of the Western North Atlantic. Novel spatial data are of scientific interest as information on the distribution, population structure, and population size of this species is limited. Additionally, we present detailed photographs and morphological data on the collected specimens. The results are discussed in relation to the dispersal abilities and population structure in syngnathids.     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

The aim of our research was to examine the ability of density gradient preparation BoviPure^? and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure^? and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure^?, but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure^? method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure^? treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure^? method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure^? could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos.     

Mycobacterium marinum Causes a Latent Infection that Can Be Reactivated by Gamma Irradiation in Adult Zebrafish

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (∼35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.     

Multi-step analysis as a tool for kinetic parameter estimation and mechanism discrimination in the reaction between tight-binding fasciculin 2 and electric eel acetylcholinesterase

The mechanism of action of a potent peptidic inhibitor fasciculin 2 (Fas2) on electric eel acetylcholinesterase (eleelAChE) has been examined in a three-level analysis. Classical steps included equilibration experiments for the evaluation of high affinity binding constant and the existence of residual hydrolytic activity in a solution of completely Fas2 saturated enzyme. The two rate constants for the association (k(on)) and the dissociation (k(off)) of Fas2 with free enzyme were determined by the time course of residual enzyme activity measurements. In the third step, with a nonclassical progress curve analysis, we found that the Fas2-enzyme complex exhibited hydrolytic activity in a butyrylcholinesterase-like kinetics. The switch appears to be a consequence of steric obstruction, but also the consequence of subtle rapid conformational changes around catalytic site, upon slow single-step binding of large Fas2 molecule at the peripheral site. An unusual unilateral effect of bound Fas2 is reflected by acylation-independent association and dissociation rates and might indeed be due to inability of small acylation agent to influence the binding of a large opponent.     

Age-Dependent Changes in the Sphingolipid Composition of Mouse CD4+ T Cell Membranes and Immune Synapses Implicate Glucosylceramides in Age-Related T Cell Dysfunction

To determine whether changes in sphingolipid composition are associated with age-related immune dysfunction, we analyzed the core sphingolipidome (i.e., all of the metabolites through the first headgroup additions) of young and aged CD4⁺ T cells. Since sphingolipids influence the biophysical properties of membranes, we evaluated the compositions of immune synapse (IS) and non-IS fractions prepared by magnetic immuno-isolation. Broadly, increased amounts of sphingomyelins, dihydrosphingomyelins and ceramides were found in aged CD4⁺ T cells. After normalizing for total sphingolipid content, a statistically significant decrease in the molar fraction of glucosylceramides was evident in both the non-IS and IS fractions of aged T cells. This change was balanced by less dramatic increases in the molar fractions of sphingomyelins and dihydrosphingomyelins in aged CD4⁺ T cells. In vitro, the direct or enzymatic enhancement of ceramide levels decreased CD4⁺ T cell proliferation without regard for the age of the responding T cells. In contrast, the in vitro inhibition of glucosylceramidase preferentially increased the proliferation of aged CD4⁺ T cells. These results suggest that reductions in glucosylceramide abundance contribute to age-related impairments in CD4⁺ T cell function.     

Assessing the radiological load on the environment in the middle Danube river basin on the basis of a study of the Kopački Rit Nature Park,Croatia

A study of the environmental radioactivity in the Kopa?ki Rit Nature Park, Croatia, is presented. This wildlife reserve is part of the Middle Danube River Basin, and it is exposed to various pollutants due to a number of human activities in the surroundings, where there is a nuclear power plant and also urban centres and areas of intense agricultural production. Results for the activity concentrations of soil and surface water samples do not indicate any elevated radioactivity level, which is confirmed by on-site measurements of ambient dose rate equivalent. An assessment of the radiological load on the local biota, carried out using the ERICA tool, implies an overall low radiological risk even if conservative values of the risk quotient are used. Therefore, human activities do not have a significant effect on the radiological load on the Kopa?ki Rit area. A similar conclusion might be made with regard to numerous similar environments in the Middle Danube River Basin.