Arabidopsis CONSTANS-LIKE3 is a positive regulator of red light signaling and root growth

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is an E3 ubiquitin ligase that represses photomorphogenesis in the dark. Therefore, proteins interacting with COP1 could be important regulators of light-dependent development. Here, we identify CONSTANS-LIKE3 (COL3) as a novel interaction partner of COP1. A green fluorescent protein-COL3 fusion protein colocalizes with COP1 to nuclear speckles when transiently expressed in plant cells. This localization requires the B-box domains in COL3, indicating a novel function of this domain. A loss-of-function col3 mutant has longer hypocotyls in red light and in short days. Unlike constans, the col3 mutant flowers early and shows a reduced number of lateral branches in short days. The mutant also exhibits reduced formation of lateral roots. The col3 mutation partially suppresses the cop1 and deetiolated1 (det1) mutations in the dark, suggesting that COL3 acts downstream of both of these repressors. However, the col3 mutation exerts opposing effects on cop1 and det1 in terms of lateral roots and anthocyanin accumulation, suggesting that COL3 also has activities that are independent of COP1 and DET1. In conclusion, we have identified COL3 as a positive regulator of photomorphogenesis that acts downstream of COP1 but can promote lateral root development independently of COP1 and also function as a daylength-sensitive regulator of shoot branching.     

Physiological routes from intra-uterine seminal contents to advancement of ovulation

Whole boar semen or seminal plasma has been demonstrated to advance the time of ovulation in gilts. As a means of clarifying this influence, the contribution of uterine lymphatics and their white cell populations has been examined. After duct visualisation with Evan's blue, lymph was sampled from a mesometrial vessel in eight pre-ovulatory gilts whose uterine lumen was infused simultaneously with whole semen in one ligated horn and saline in the contralateral ligated horn. Lymph was collected from cannulated vessels for periods of up to four hours under general anaesthesia. Thereafter, mesometrial lymph nodes, utero-tubal junction and uterine wall tissues were sampled. The proportion of nucleated cells in the sampled lymph increased towards the end of the collection period, but erythrocytes were found in all instances preventing a meaningful differentiation and identification of leukocytes. Prominent uterine lymph nodes were present in the mesometrium on both sides of the reproductive tract in 7 of 10 gilts. Differences in cellular contents were demonstrated between the side of the tract infused with semen and that infused with saline control. Two of 4 gilts had lower values for CD4 (Cluster Differentiation) and 3 of 6 gilts higher values for MHC II (Major Histocompatibility Complex) markers on the side challenged with semen. In contrast, values remained constant for CD8 but ranged widely for CD18. Immunohistochemical analysis of uterine tissue samples for MHC II+ cells revealed significant differences (P < 0.05) between the control and semen-treated ligated portions of the horns, as well as between the tissue sample of uterine wall and that from the utero-tubal junction, but there were no significant differences for CD4+ cells. It therefore remains plausible that semen-induced cytokines in the uterine lymph undergo counter-current transfer to the ipsilateral ovary and accelerate the final maturation of pre-ovulatory Graafian follicles.     

Studying the uptake of cell-penetrating peptides

More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.     

Active induction of experimental allergic encephalomyelitis

This protocol details a method to actively induce experimental allergic encephalomyelitis (EAE), a widely used animal model for studies of multiple sclerosis. EAE is induced by stimulating T-cell-mediated immunity to myelin antigens. Active induction of EAE is accomplished by immunization with myelin antigens emulsified in adjuvant. This protocol focuses on induction of EAE in mice; however, the same principles apply to EAE induction in other species. EAE in rodents is manifested typically as ascending flaccid paralysis with inflammation targeting the spinal cord. However, more diverse clinical signs can occur in certain strain/antigen combinations in rodents and in other species, reflecting increased inflammation in the brain.     

Passive induction of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a widely used animal model of the human demyelinating disease multiple sclerosis. EAE is initiated by immunization with myelin antigens in adjuvant or by adoptive transfer of myelin-specific T cells, resulting in inflammatory infiltrates and demyelination in the central nervous system. Induction of EAE in rodents typically results in ascending flaccid paralysis with inflammation primarily targeting the spinal cord. This protocol describes passive induction of EAE by adoptive transfer of T cells isolated from mice primed with myelin antigens into na?ve mice. The advantages of using this method versus active induction of EAE are discussed.     

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L)

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens′, was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.     

Genome Sequence of Lactobacillus crispatus ST1

Lactobacillus crispatus is a common member of the beneficial microbiota present in the vertebrate gastrointestinal and human genitourinary tracts. Here, we report the genome sequence of L. crispatus ST1, a chicken isolate displaying strong adherence to vaginal epithelial cells.Lactobacillus crispatus can persist in the vertebrate gastrointestinal tract and is among the most prevalent species of the Lactobacillus-dominated human vaginal microbiota (2, 9, 13, 14). It belongs to the so-called acidophilus group (3), which has attracted interest because some of its species are important factors in the production of fermented foods (12) and some can, at least transiently, colonize the human host (2, 9, 13, 14). Moreover, some specific strains, mainly L. acidophilus NCFM and L. johnsonii NCC 533, have received prominence as intestinal-health-promoting microbes (4). Although the genomes of seven members of the acidophilus complex have been sequenced to date (12), the genome sequences of L. crispatus and other predominant lactobacillar species in the urogenital flora have mostly remained obscure. Vaginal lactobacilli can have an important role in controlling the health of the host (2, 14). They can, for example, positively influence and stabilize the host''s vaginal microbiota via the production of compounds that are acidic or exert a direct inhibiting action toward pathogenic bacteria (2, 14). In addition to the antimicrobial compounds, the competitive exclusion of pathogens is another mechanism by which the host''s microbiota can be balanced (2). L. crispatus ST1 was originally isolated from the crop of a chicken, and PCR profiling of L. crispatus isolates has verified it to be an abundant colonizer of the chicken crop (6, 8). It also displays a strong protein-dependent adhesion to the epithelial cells of the human vagina and has been shown to inhibit the adhesion of avian pathogenic Escherichia coli (6, 7).The genome was sequenced (18× coverage) using a 454 pyrosequencer with GS FLX chemistry (Roche). The contig order was confirmed and gaps were filled by sequencing PCR fragments from the genomic DNA template using ABI 3730 and Big Dye chemistry (Applied Biosystems). Genomic data were processed using the Staden Package (11) and gsAssembler (Roche). Coding sequences (CDSs) were predicted using Glimmer3 (5) followed by manual curation of the start sites. The remaining intergenic regions were reanalyzed for missed CDSs by using BlastX (1). Annotation transfer was performed based on a BlastP search, followed by Blannotator analysis using default settings (http://ekhidna.biocenter.helsinki.fi/poxo/blannotator) and manual verification. Orthologous groups between the different lactobacillar proteomes were identified using OrthoMCL (10).The genome of L. crispatus ST1 consists of a single circular chromosome 2.04 Mbp in size, with an overall G+C content of 37%, without any plasmids. There are 64 tRNA genes, 4 rRNA operons, and 2 CRISPR loci. Out of the 2,024 predicted CDSs, a putative function was assigned to 77%, whereas 10% of the CDSs were annotated as conserved and 13% as novel. Based on the orthologous grouping, 302 (15%) of the CDSs encoded by ST1 have no detectable homologs in any of the Lactobacillus proteomes published to date.     

Nuclear Receptor Liver X Receptor Is O-GlcNAc-modified in Response to Glucose

Post-translational modification of nucleocytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has for the last 25 years emerged as an essential glucose-sensing mechanism. The liver X receptors (LXRs) function as nutritional sensors for cholesterol-regulating lipid metabolism, glucose homeostasis, and inflammation. LXRs are shown to be post-translationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively. In the present study, we show for the first time that LXRα and LXRβ are targets for glucose-hexosamine-derived O-GlcNAc modification in human Huh7 cells. Furthermore, we observed increased hepatic LXRα O-GlcNAcylation in vivo in refed mice and in streptozotocin-induced refed diabetic mice. Importantly, induction of LXRα O-GlcNAcylation in both mouse models was concomitant with increased expression of the lipogenic gene SREBP-1c (sterol regulatory element-binding protein 1c). Furthermore, glucose increased LXR/retinoic acid receptor-dependent activation of luciferase reporter activity driven by the mouse SREBP-1c promoter via the hexosamine biosynthetic pathway in Huh7 cells. Altogether, our results suggest that O-GlcNAcylation of LXR is a novel mechanism by which LXR acts as a glucose sensor affecting LXR-dependent gene expression, substantiating the crucial role of LXR as a nutritional sensor in lipid and glucose metabolism.