Influence of the effector peptide of MARCKS-related protein on actin polymerization: a kinetic analysis

The members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with membranes, protein kinase C, and calmodulin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. This domain is also involved in the interaction between MARCKS/MRP and actin. In this article we show that a peptide corresponding to the effector domain of MRP, the effector peptide, strongly influences the dynamics of actin polymerization. Depending on the stoichiometric ratio of effector peptide to actin the peptide either accelerates or retards the actin polymerization process, which takes place in the presence of near-physiological salt concentrations. A model is developed in which this phenomenon is explained by two independent nucleation processes involving free actin monomers and peptide-bound actin monomers, respectively. As a control, a possible regulatory mechanism has been investigated: we show that calmodulin inhibits the actin polymerizing activity of the MRP effector peptide, thereby validating our model approach.     

Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system

alpha-Synuclein (alpha-Syn) is a 14 kDa protein of unknown function that has been implicated in the pathophysiology of Parkinson's disease (PD). Here, we show that alpha-Syn-/- mice are viable and fertile, exhibit intact brain architecture, and possess a normal complement of dopaminergic cell bodies, fibers, and synapses. Nigrostriatal terminals of alpha-Syn-/- mice display a standard pattern of dopamine (DA) discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli that can be mimicked by elevated Ca2+. Concurrent with the altered DA release, alpha-Syn-/- mice display a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine. These findings support the hypothesis that alpha-Syn is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission.     

Collagen distribution and expression of collagen-binding alpha1beta1 (VLA-1) and alpha2beta1 (VLA-2) integrins on CD4 and CD8 T cells during influenza infection

Most viral infections occur in extralymphoid tissues, yet the mechanisms that regulate lymphocytes in these environments are poorly understood. One feature common to many extralymphoid environments is an abundance of extracellular matrix. We have studied the expression of two members of the beta(1) integrin family of collagen-binding receptors, alpha(1)beta(1) and alpha(2)beta(1) (CD49a, VLA-1 and CD49b, VLA-2, respectively), on CD4 and CD8 T cells during the response to influenza infection in the lung. Flow cytometry showed that whereas T cells infiltrating the lung and airways can express both CD49a and CD49b, CD49a expression was most strongly associated with the CD8+ subset. Conversely, though fewer CD4+ T cells expressed CD49a, most CD4+ cells in the lung tissue or airways expressed CD49b. This reciprocal pattern suggested that CD4 and CD8 T cells might localize differently within the lung tissue and this was supported by immunofluorescent analysis. CD8+ cells tended to localize in close proximity to the collagen IV-rich basement membranes of either the airways or blood vessels, whereas CD4+ cells tended to localize in the collagen I-rich interstitial spaces, with few in the airways. These observations suggest that CD4 T cell interaction with the tissue microenvironment is distinct from CD8 T cells and support the concept that CD4+ T cells in peripheral tissues are regulated differently than the CD8 subset.     

The catalysis of the 1,1-proton transfer by alpha-methyl-acyl-CoA racemase is coupled to a movement of the fatty acyl moiety over a hydrophobic, methionine-rich surface

The crystal structure of a novel fungal lectin from Sclerotium rolfsii (SRL) in its free form and in complex with N-acetyl-d-galactosamine (GalNAc) and N-acetyl- d -glucosamine (GlcNAc) has been determined at 1.1 A, 2.0 A, and 1.7 A resolution, respectively. The protein structure is composed of two beta-sheets, which consist of four and six beta-strands, connected by two alpha-helices. Sequence and structural comparisons reveal that SRL is the third member of a newly identified family of fungal lectins, which includes lectins from Agaricus bisporus and Xerocomus chrysenteron that share a high degree of structural similarity and carbohydrate specificity. The data for the free SRL are the highest resolution data for any protein of this family. The crystal structures of the SRL in complex with two carbohydrates, GalNAc and GlcNAc, which differ only in the configuration of a single epimeric hydroxyl group, provide the structural basis for its carbohydrate specificity. SRL has two distinct carbohydrate-binding sites, a primary and a secondary. GalNAc binds at the primary site, whereas GlcNAc binds only at the secondary site. Thus, SRL has the ability to recognize and probably bind at the same time two different carbohydrate structures. Structural comparison to Agaricus bisporus lectin-carbohydrate complexes reveals that the primary site is also able to bind the Thomsen-Friedenreich antigen (Galbeta1-->3GalNAc-alpha- glycan structures) whereas the secondary site cannot. The features of the molecular recognition at the two sites are described in detail.     

Assessing the Risk of Herbicides to Terrestrial Non-Target Plants Using Higher-Tier Studies

Risk assessment for non-target plants is based on single species phytotoxicity tests. This approach may not reflect relevant ecological processes in terrestrial ecosystems. The current risk assessment scheme is based on endpoints measured at the species level and the assessment of ecological effects relies on the extrapolation from one species to another or from a single species to a community. This extrapolation contains many uncertainties that may be reduced by adopting more realistic testing approaches. However, currently higher-tier plant studies are not obligatory in herbicide risk assessment. We reviewed the published literature and found that potential higher-tier approaches for terrestrial non-target plants are extremely limited. Sixteen studies were found that assessed the effects of herbicides on non-target plants by performing microcosms, mesocosms, or field studies. These studies showed that microcosms might provide useful data and help to reduce uncertainties associated with single-species tests. However, due to the limited number of available studies, much work is required to develop appropriate testing methods for regulatory processes. In addition, field experiments are necessary to establish baseline knowledge concerning the effects of herbicides on natural plant communities and to compare data generated in tiered testing approaches with data obtained from natural systems.     

Complete genome sequence of the Clostridium difficile laboratory strain 630Δerm reveals differences from strain 630, including translocation of the mobile element CTn5

Background

Clostridium difficile strain 630Δerm is a spontaneous erythromycin sensitive derivative of the reference strain 630 obtained by serial passaging in antibiotic-free media. It is widely used as a defined and tractable C. difficile strain. Though largely similar to the ancestral strain, it demonstrates phenotypic differences that might be the result of underlying genetic changes. Here, we performed a de novo assembly based on single-molecule real-time sequencing and an analysis of major methylation patterns.

Results

In addition to single nucleotide polymorphisms and various indels, we found that the mobile element CTn5 is present in the gene encoding the methyltransferase rumA rather than adhesin CD1844 where it is located in the reference strain.

Conclusions

Together, the genetic features identified in this study may help to explain at least part of the phenotypic differences. The annotated genome sequence of this lab strain, including the first analysis of major methylation patterns, will be a valuable resource for genetic research on C. difficile.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1252-7) contains supplementary material, which is available to authorized users.     

Evolutionary conservation of the FLOWERING LOCUS C-mediated vernalization response: evidence from the sugar beet (Beta vulgaris)

In many plant species, exposure to a prolonged period of cold during the winter promotes flowering in the spring, a process termed vernalization. In Arabidopsis thaliana, the vernalization requirement of winter-annual ecotypes is caused by the MADS-box gene FLOWERING LOCUS C (FLC), which is a repressor of flowering. During the vernalization process, FLC is downregulated by alteration of its chromatin structure, thereby permitting flowering to occur. In wheat, a vernalization requirement is imposed by a different repressor of flowering, suggesting that some components of the regulatory network controlling the vernalization response differ between monocots and dicots. The extent to which the molecular mechanisms underlying vernalization have been conserved during the diversification of the angiosperms is not well understood. Using phylogenetic analysis, we identified homologs of FLC in species representing the three major eudicot lineages. FLC homologs have not previously been documented outside the plant family Brassicaceae. We show that the sugar beet FLC homolog BvFL1 functions as a repressor of flowering in transgenic Arabidopsis and is downregulated in response to cold in sugar beet. Cold-induced downregulation of an FLC-like floral repressor may be a central feature of the vernalization response in at least half of eudicot species.