Dihydrotestosterone modulates spatial working-memory performance in male mice

Androgens affect cognitive processes in both humans and animals. The effects of androgens may be limited to certain cognitive domains, specifically spatial memory, but this hypothesis remains elusive. Here, we tested castrated and sham-operated mice in various behavioral tasks to ask whether androgens affect multiple or specific cognitive domains in male mice. Castration impaired spatial working memory performance in the delayed matching to place water maze task following a 1-h, but not a 1-min, retention interval, as has been reported for rats. In contrast, castration had no effect on novel object recognition memory, spatial reference memory in the water maze, motor coordination, or passive avoidance memory. Castration increased anxiety-like behavior in the open field test, but not the elevated zero maze. Finally, we assessed the effects of androgen replacement with non-aromatizable dihydrotestosterone on spatial working memory following various retention intervals. Dihydrotestosterone recovered spatial memory performance following a 24-h, but not a 1-h retention interval, and had no effect at other retention intervals. These data support that in male mice androgens specifically affect spatial working memory performance, and that the neurobiological processes underlying spatial memory formation may be differentially affected by androgens.     

The cin and rai Quorum-Sensing Regulatory Systems in Rhizobium leguminosarum Are Coordinated by ExpR and CinS,a Small Regulatory Protein Coexpressed with CinI

To understand how the Rhizobium leguminosarum raiI-raiR quorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-made N-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required for raiR expression, and RaiR is required to induce raiI. Since raiR (and raiI) expression is also reduced in cinI and cinR quorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induce raiR. However, added CinI-made AHLs did not induce raiR expression in a cinI mutant. The reduced raiR expression in cinI and cinR mutants was due to lack of expression of cinS immediately downstream of cinI. cinS encodes a 67-residue protein, translationally coupled to CinI, and cinS acts downstream of expR for raiR induction. Cloned cinS in R. leguminosarum caused an unusual collapse of colony structure, and this was delayed by mutation of expR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations in cinI, cinR, cinS, and expR all reduced expression of plyB, encoding an EPS glycanase, and mutation of plyB abolished the effect of cloned cinS on colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, including Rhizobium etli; the previously observed effect of cinI and cinR mutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs.Production of N-acyl homoserine lactones (AHLs) is common to many plant-associated bacteria (7), in which it is usually associated with population density-dependent regulation of genes affecting adaptive responses (49). Within the family Rhizobiaceae, population density-regulated gene expression (quorum sensing) mediated via AHLs has been identified in several agrobacteria and rhizobia (13, 51). In Agrobacterium spp., quorum-sensing regulation was initially identified as a mechanism of regulating plasmid transfer. As the bacterial population density increases, plasmid transfer genes are induced by TraR in response to AHLs made by TraI (55). In several rhizobia, traI-like AHL synthase genes are also in an operon along with plasmid transfer genes (13).There are other quorum-sensing loci in different strains of rhizobia. In Sinorhizobium meliloti strain Rm1021, AHLs produced by SinI activate SinR and ExpR, LuxR-type regulators, to induce several genes, including those determining the production of an exopolysaccharide, exopolysaccharide II (EPS-II) (17, 23, 24, 35), that plays an important role in the symbiosis. In S. meliloti, two LuxR-type regulators, VisN and VisR, are involved in chemotaxis and motility (24, 44). Rhizobium etli has multiple AHL synthase genes (9, 39), but the functions of many of the regulated genes remain to be established. The cinR and cinI genes are required for normal symbiotic nitrogen fixation and swarming in R. etli (5, 9, 11) and for normal levels of expression of raiI, which encodes another AHL synthase. The expression of raiI in R. etli is regulated by RaiR (39).Analysis of AHLs produced by strain A34 of Rhizobium leguminosarum bv. viciae led to the characterization of four LuxI-type AHL synthases (RhiI, CinI, RaiI, and TraI) and five LuxR-type regulators (RhiR, CinR, RaiR, TraR, and BisR) (8, 31, 50, 53). In this strain, the cinI and cinR genes are chromosomally located; CinI produces N-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3-OH-C_14:1-HSL) (20, 31), CinR induces cinI expression in response to this AHL (31), and this appears to be associated with adaptation to starvation and salt stress (47). Mutation of cinI or cinR affects the expression of the other three AHL synthase genes in R. leguminosarum bv. viciae strain A34. Thus, in a cinI mutant, the expression of raiI is reduced, resulting in very low levels of 3-OH-C₈-HSL, the major AHL made by RaiI (53). Similarly, the expression levels of the traI and rhiI genes on the symbiotic plasmid pRL1JI are reduced in cinI and cinR mutants (31). RhiI-made AHLs activate RhiR to induce the expression of the rhiABC operon in R. leguminosarum bv. viciae (38), enhancing the interaction with the legume host (8).The cinI and cinR quorum-sensing genes control induction of the traI and traR quorum-sensing regulons via CinI-made 3-OH-C_14:1-HSL, which activates BisR (another LuxR-type regulator) to induce traR and hence traI (12). However, the mechanism by which cinI and/or cinR control raiI and raiR expression has not been established. In this work we demonstrate that raiI and raiR expression requires both expR and a small gene (cinS) cotranscribed with cinI. CinS also regulates the expression of plyB encoding an extracellular glycanase and is required for swarming of R. etli.     

Prolectin, a Glycan-binding Receptor on Dividing B Cells in Germinal Centers

Prolectin, a previously undescribed glycan-binding receptor, has been identified by re-screening of the human genome for genes encoding proteins containing potential C-type carbohydrate-recognition domains. Glycan array analysis revealed that the carbohydrate-recognition domain in the extracellular domain of the receptor binds glycans with terminal α-linked mannose or fucose residues. Prolectin expressed in fibroblasts is found at the cell surface, but unlike many glycan-binding receptors it does not mediate endocytosis of a neoglycoprotein ligand. However, compared with other known glycan-binding receptors, the receptor contains an unusually large intracellular domain that consists of multiple sequence motifs, including phosphorylated tyrosine residues, that allow it to interact with signaling molecules such as Grb2. Immunohistochemistry has been used to demonstrate that prolectin is expressed on a specialized population of proliferating B cells in germinal centers. Thus, this novel receptor has the potential to function in carbohydrate-mediated communication between cells in the germinal center.Membrane-bound mammalian glycan-binding receptors, often referred to as lectins, are believed to play multiple distinct roles in the immune system, decoding information in complex oligosaccharide structures on cell surfaces and soluble glycoproteins (1, 2). A host of glycan-binding receptors on dendritic cells and macrophages function in pathogen recognition, often resulting in uptake of microbes through endocytic mechanisms. Examples include the mannose receptor, DC-SIGN,³ langerin, and the macrophage galactose receptor. Glycan-binding receptors can also recognize glycans found on the surfaces of mammalian cells. Some of these receptors, such as the selectins, mediate adhesion between leukocytes and endothelia (3, 4). A small number of receptors, notably members of the siglec family, bind mammalian-type glycans and have been shown to have potential signaling functions (5). While multiple glycan-binding receptors have been described on cells of the myeloid lineage, the complement of such receptors on lymphocytes is much more restricted. The best characterized examples are the T-cell adhesion molecule L-selectin (4) and the B-cell receptor CD22, also designated siglec-2 (5).Genomic screening for potential glycan-binding receptors has usually been undertaken by initially searching for the presence of one of the several types of structural domains that are known to support sugar-binding activity (6). Knowledge of the structures of multiple families of modular carbohydrate-recognition domains (CRDs) has facilitated identification of proteins with potential sugar-binding activity and can lead to predictions of what types of ligands might be bound. Although the human genome has been extensively screened with profile-recognition algorithms that identify common sequence motifs associated with CRDs, refinements to the genome sequence and improvements in gene-recognition algorithms occasionally result in detection of novel proteins that contain putative CRDs.We describe a previously undetected glycan-binding receptor identified by re-screening of the human genome and provide characterization of its molecular and cellular properties. Based on its expression in a specialized population of proliferating B cells in germinal centers, we propose that it be designated prolectin. Our results suggest that prolectin functions in carbohydrate-mediated communication between cells in the germinal center.     

A Multiplex Human Syndrome Implicates a Key Role for Intestinal Cell Kinase in Development of Central Nervous, Skeletal, and Endocrine Systems

Six infants in an Old Order Amish pedigree were observed to be affected with endocrine-cerebro-osteodysplasia (ECO). ECO is a previously unidentified neonatal lethal recessive disorder with multiple anomalies involving the endocrine, cerebral, and skeletal systems. Autozygosity mapping and sequencing identified a previously unknown missense mutation, R272Q, in ICK, encoding intestinal cell kinase (ICK). Our results established that R272 is conserved across species and among ethnicities, and three-dimensional analysis of the protein structure suggests protein instability due to the R272Q mutation. We also demonstrate that the R272Q mutant fails to localize at the nucleus and has diminished kinase activity. These findings suggest that ICK plays a key role in the development of multiple organ systems.     

Vancomycin and Oritavancin Have Different Modes of Action in Enterococcus faecium

The increasing frequency of Enterococcus faecium isolates with multidrug resistance is a serious clinical problem given the severely limited number of therapeutic options available to treat these infections. Oritavancin is a promising new alternative in clinical development that has potent antimicrobial activity against both staphylococcal and enterococcal vancomycin-resistant pathogens. Using solid-state NMR to detect changes in the cell-wall structure and peptidoglycan precursors of whole cells after antibiotic-induced stress, we report that vancomycin and oritavancin have different modes of action in E. faecium. Our results show the accumulation of peptidoglycan precursors after vancomycin treatment, consistent with transglycosylase inhibition, but no measurable difference in cross-linking. In contrast, after oritavancin exposure, we did not observe the accumulation of peptidoglycan precursors. Instead, the number of cross-links is significantly reduced, showing that oritavancin primarily inhibits transpeptidation. We propose that the activity of oritavancin is the result of a secondary binding interaction with the E. faecium peptidoglycan. The hypothesis is supported by results from ¹³C{¹⁹F} rotational-echo double-resonance (REDOR) experiments on whole cells enriched with l-[1-¹³C]lysine and complexed with desleucyl [¹⁹F]oritavancin. These experiments establish that an oritavancin derivative with a damaged d-Ala-d-Ala binding pocket still binds to E. faecium peptidoglycan. The ¹³C{¹⁹F} REDOR dephasing maximum indicates that the secondary binding site of oritavancin is specific to nascent and template peptidoglycan. We conclude that the inhibition of transpeptidation by oritavancin in E. faecium is the result of the large number of secondary binding sites relative to the number of primary binding sites.     

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Nuclear factor (NF)-κB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-κB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65^−/− murine fibroblasts immortalize at considerably faster rates than RelA/p65^+/+ cells. The ability of RelA/p65^−/− fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65^−/− fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-κB. Altogether, our findings present a fresh perspective on the role of NF-κB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.