Conserved linkage of two genes on the same macronuclear chromosome in spirotrichous ciliates

Macronuclear chromosomes of spirotrichous ciliates are mainly “nanochromosomes” containing only a single gene. We identified a two-gene chromosome in the spirotrich Sterkiella histriomuscorum (formerly Oxytricha trifallax) which, unlike other characterized two-gene molecules, contains reading frames oriented tail to tail. These are homologs of ribosomal protein L29 (RPL29) and cyclophilin. We found that both genes are transcribed, with their polyadenylation sites on opposite strands separated by only 135 bp. Furthermore, both genes in S. histriomuscorum are present only on one macronuclear chromosome and do not occur alone or linked to other genes. The corresponding micronuclear locus is fragmented into three nonscrambled gene segments (MDSs), separated by two noncoding segments (IESs). We also found that these two genes are linked on a macronuclear chromosome, similarly arranged tail to tail, in the three spirotrichs Stylonychia lemnae, Uroleptus sp., and Holosticha sp.. In addition, single-gene macronuclear chromosomes containing only the RPL29 gene were detected in the earlier diverged Holosticha and Uroleptus. These observations suggest a possible evolutionary trend towards loss of chromosomal breakage between these two genes. This study is the first to examine gene linkage in the macronucleus of several spirotrichs and may provide insight into the evolution of multi-gene macronuclear chromosomes and chromosomal fragmentation in spirotrichs. Electronic Supplementary Material Supplementary material is available for this article at     

Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana

The X-ray crystal structure of the At5g18200.1 protein has been determined to a nominal resolution of 2.30 A. The structure has a histidine triad (HIT)-like fold containing two distinct HIT-like motifs. The sequence of At5g18200.1 indicates a distant family relationship to the Escherichia coli galactose-1-P uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein confirms this relationship. The At5g18200.1 protein does not demonstrate GalT activity but instead catalyzes adenylyl transfer in the reaction of ADP-glucose with various phosphates. The best acceptor among those evaluated is phosphate itself; thus, the At5g18200.1 enzyme appears to be an ADP-glucose phosphorylase. The enzyme catalyzes the exchange of (14)C between ADP-[(14)C]glucose and glucose-1-P in the absence of phosphate. The steady state kinetics of exchange follows the ping-pong bi-bi kinetic mechanism, with a k(cat) of 4.1 s(-)(1) and K(m) values of 1.4 and 83 microM for ADP-[(14)C]glucose and glucose-1-P, respectively, at pH 8.5 and 25 degrees C. The overall reaction of ADP-glucose with phosphate to produce ADP and glucose-1-P follows ping-pong bi-bi steady state kinetics, with a k(cat) of 2.7 s(-)(1) and K(m) values of 6.9 and 90 microM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 degrees C. The kinetics are consistent with a double-displacement mechanism that involves a covalent adenylyl-enzyme intermediate. The X-ray crystal structure of this intermediate was determined to 1.83 A resolution and shows the AMP group bonded to His(186). The value of K(eq) in the direction of ADP and glucose-1-P formation is 5.0 at pH 7.0 and 25 degrees C in the absence of a divalent metal ion, and it is 40 in the presence of 1 mM MgCl(2).     

Combined treatment with lisofylline and exendin-4 reverses autoimmune diabetes

Type 1 diabetes mellitus (T1DM) is an autoimmune disease leading to near complete pancreatic beta-cell destruction. New evidence suggests that beta-cell regeneration is possible, but ongoing autoimmune damage prevents restoration of beta-cell mass. We tested the hypothesis that simultaneously blocking autoimmune cytokine damage and supplying a growth-promoting stimulus for beta-cells would provide a novel approach to reverse T1DM. Therefore, in this study we combined lisofylline to suppress autoimmunity and exendin-4 to enhance beta-cell proliferation for treating autoimmune-mediated diabetes in the non-obese diabetic (NOD) mouse model. We found that this combined therapy effectively reversed new-onset diabetes within a week of therapy, and even maintained euglycemia up to 145 days after treatment withdrawal. The therapeutic effect of this regimen was associated with improved beta-cell metabolism and insulin secretion, while reducing beta-cell apoptosis. It is possible that such combined therapy could become a new strategy to defeat T1DM in humans.     

Interleukin-10 does not mediate inhalational tolerance in a chronic model of ovalbumin-induced allergic airway disease

OBJECTIVE: IL-10 is a potent anti-inflammatory cytokine, and IL-10-producing regulatory T cells are effective inhibitors of murine asthmatic responses. This study determined whether IL-10-dependent mechanisms mediated the local inhalational tolerance seen with chronic inhalational exposure to antigen. METHODS: Wildtype and IL-10(-/-) mice were sensitized with ovalbumin (OVA) and then challenged with daily OVA inhalations for 10 days or 6 weeks. RESULTS: The 10-day animals developed allergic airway disease, characterized by BAL eosinophilia, histologic airway inflammation and mucus secretion, methacholine hyperresponsiveness, and OVA-specific IgE production. These changes were more pronounced in IL-10(-/-) mice. The 6-week IL-10(-/-) and wildtype animals both developed inhalational tolerance, with resolution of airway inflammation but persistence of OVA-specific IgE production. CONCLUSION: IL-10 may have anti-inflammatory effects in the acute stage of murine allergic airways disease, but the cytokine does not mediate the development of local inhalational tolerance with chronic antigen exposure.     

Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation

Mutations in fibroblast growth factor receptors (Fgfrs) 1-3 cause skeletal disease syndromes in humans. Although these Fgfrs are expressed at various stages of chondrocyte and osteoblast development, their function in specific skeletal cell types is poorly understood. Using conditional inactivation of Fgfr1 in osteo-chondrocyte progenitor cells and in differentiated osteoblasts, we provide evidence that FGFR1 signaling is important for different stages of osteoblast maturation. Examination of osteogenic markers showed that inactivation of FGFR1 in osteo-chondro-progenitor cells delayed osteoblast differentiation, but that inactivation of FGFR1 in differentiated osteoblasts accelerated differentiation. In vitro osteoblast cultures recapitulated the in vivo effect of FGFR1 on stage-specific osteoblast maturation. In immature osteoblasts, FGFR1 deficiency increased proliferation and delayed differentiation and matrix mineralization, whereas in differentiated osteoblasts, FGFR1 deficiency enhanced mineralization. Furthermore, FGFR1 deficiency in differentiated osteoblasts resulted in increased expression of Fgfr3, a molecule that regulates the activity of differentiated osteoblasts. Mice lacking Fgfr1, either in progenitor cells or in differentiated osteoblasts, showed increased bone mass as adults. These data demonstrate that signaling through FGFR1 in osteoblasts is necessary to maintain the balance between bone formation and remodeling through a direct effect on osteoblast maturation.     

Regulation of neuronal lineage decisions by the HES-related bHLH protein REF-1

Members of the HES subfamily of bHLH proteins play crucial roles in neural patterning via repression of neurogenesis. In C. elegans, loss-of-function mutations in ref-1, a distant nematode-specific member of this subfamily, were previously shown to cause ectopic neurogenesis from postembryonic lineages. However, while the vast majority of the nervous system in C. elegans is generated embryonically, the role of REF-1 in regulating these neural lineage decisions is unknown. Here, we show that mutations in ref-1 result in the generation of multiple ectopic neuron types derived from an embryonic neuroblast. In wild-type animals, neurons derived from this sublineage are present in a left/right symmetrical manner. However, in ref-1 mutants, while the ectopically generated neurons exhibit gene expression profiles characteristic of neurons on the left, they are present only on the right side. REF-1 functions in a Notch-independent manner to regulate this ectopic lineage decision. We also demonstrate that loss of REF-1 function results in defective differentiation of an embryonically generated serotonergic neuron type. These results indicate that REF-1 functions in both Notch-dependent and independent pathways to regulate multiple developmental decisions in different neuronal sublineages.     

The Drosophila boundary element-associated factors BEAF-32A and BEAF-32B affect chromatin structure

Binding sites for the Drosophila boundary element-associated factors BEAF-32A and -32B are required for the insulator activity of the scs' insulator. BEAF binds to hundreds of sites on polytene chromosomes, indicating that BEAF-utilizing insulators are an important class in Drosophila. To gain insight into the role of BEAF in flies, we designed a transgene encoding a dominant-negative form of BEAF under GAL4 UAS control. This BID protein encompasses the BEAF self-interaction domain. Evidence is provided that BID interacts with BEAF and interferes with scs' insulator activity and that BEAF is the major target of BID in vivo. BID expression during embryogenesis is lethal, implying that BEAF is required during early development. Expression of BID in eye imaginal discs leads to a rough-eye phenotype, and this phenotype is rescued by a third copy of the BEAF gene. Expression of BID in salivary glands leads to a global disruption of polytene chromatin structure, and this disruption is largely rescued by an extra copy of BEAF. BID expression also enhances position-effect variegation (PEV) of the w(m4h) allele and a yellow transgene inserted into the pericentric heterochromatin of chromosome 2R, while a third copy of the BEAF gene suppresses PEV of both genes. These results support the hypothesis that BEAF-dependent insulators function by affecting chromatin structure or dynamics.     

Context-dependent intergenerational effects: the interaction between past and present environments and its effect on population dynamics

Intergenerational effects arise when parents' actions influence the reproduction and survival of their offspring and possibly later descendants. Models suggest that intergenerational effects have important implications for both population dynamical patterns and the evolution of life-history traits. However, these will depend on the nature and duration of intergenerational effects. Here we show that manipulating parental food environments of soil mites produced intergenerational effects that were still detectable in the life histories of descendents three generations later. Intergenerational effects varied in different environments and from one generation to the next. In low-food environments, variation in egg size altered a trade-off between age and size at maturity and had little effect on the size of eggs produced in subsequent generations. Consequently, intergenerational effects decreased over time. In contrast, in high-food environments, variation in egg size predominantly influenced a trade-off between fecundity and adult survival and generated increasing variation in egg size. As a result, the persistence and significance of intergenerational effects varied between high- and low-food environments. Context-dependent intergenerational effects can therefore have complex but important effects on population dynamics.     

Bacterial production of biologically active canine interleukin-1beta by seamless SUMO tagging and removal

Interleukin 1beta (IL-1beta) is a potent stimulator of extracellular matrix degradation in models of osteoarthritis (OA). In contrast to bovine explant models which effectively respond to recombinant human IL-1beta, canine models are relatively refractory to human IL-1beta stimulation. Canine IL-1beta cDNA was cloned in order to produce a fully potent species matched preparation of IL-1beta for use specifically in canine models of OA. Established methods for the production of various orthologous IL-1beta proteins from different species are problematic due to the exquisite sensitivity of the mature IL-1beta product to N-terminal variations and the intrinsic technical challenges associated with producing an unmodified product. We have applied a seamless method of SUMO tagging and removal in order to produce a homogeneous unmodified preparation of canine IL-1beta from Escherichia coli which was found to be a potent inducer of aggrecanase activity in isolated canine articular chondrocytes. This method combines highly efficient aspects of seamless plasmid engineering, protein purification, and precise tag removal.