Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks

Loss or addition of nucleotides at junctions generated by V(D)J recombination significantly expands the antigen-receptor repertoire. Addition of nontemplated (N) nucleotides is carried out by terminal deoxynucleotidyl transferase (TdT), whose only known physiological role is to create diversity at V(D)J junctions during lymphocyte development. Although purified TdT can act at free DNA ends, its ability to add nucleotides (i.e. form N regions) at coding joints appears to depend on the nonhomologous end-joining factor Ku80. Because the DNA ends generated during V(D)J rearrangements remain associated with the RAG proteins after cleavage, TdT might be targeted for N region addition through interactions with RAG proteins or with Ku80 during remodeling of the post-cleavage complex. Such regulated access would help to prevent TdT from acting at other types of broken ends and degrading the fidelity of end joining. To test this hypothesis, we measured TdT’s ability to add nucleotides to endonuclease-induced chromosomal and extrachromosomal breaks. In both cases TdT added nucleotides efficiently to the cleaved DNA ends. Strikingly, the frequency of N regions at non-V(D)J-generated ends was not dependent on Ku80. Thus our results suggest that Ku80 is required to allow TdT access to RAG post-cleavage complexes, providing support for the hypothesis that Ku is involved in disassembling or remodeling the post-cleavage complex. We also found that N regions were abnormally long in the absence of Ku80, indicating that Ku80 may regulate TdT’s activity at DNA ends in vivo.     

Role of Wnt-5a in the Determination of Human Mesenchymal Stem Cells into Preadipocytes

Increasing adipocyte size as well as numbers is important in the development of obesity and type 2 diabetes, with adipocytes being generated from mesenchymal precursor cells. This process includes the determination of mesenchymal stem cells (MSC) into preadipocytes (PA) and the differentiation of PA into mature fat cells. Although the process of differentiation has been highly investigated, the determination in humans is poorly understood. In this study, we compared human MSC and human committed PA on a cellular and molecular level to gain further insights into the regulatory mechanisms in the determination process. Both cell types showed similar morphology and expression patterns of common mesenchymal and hematopoietic surface markers. However, although MSC were able to differentiate into adipocytes and osteocytes, PA were only able to undergo adipogenesis, indicating that PA lost their multipotency during determination. WNT-5a expression showed significantly higher levels in MSC compared with PA suggesting that WNT-5a down-regulation might be important in the determination process. Indeed, incubation of human MSC in medium containing neutralizing WNT-5a antibodies abolished their ability to undergo osteogenesis, although adipogenesis was still possible. An opposite effect was achieved using recombinant WNT-5a protein. On a molecular level, WNT-5a was found to promote c-Jun N-terminal kinase-dependent intracellular signaling in MSC. Activation of this noncanonical pathway resulted in the induction of osteopontin expression further indicating pro-osteogenic effects of WNT-5a. Our data suggest that WNT-5a is necessary to maintain osteogenic potential of MSC and that inhibition of WNT-5a signaling therefore plays a role in their determination into PA in humans.     

Dietary preference for methionine sources in weaned pigs

The objective of the current study was to investigate the preference of weaned pigs given the choice of diets supplemented with dl-methionine (DLM) or liquid dl-methionine hydroxy analog-free acid (MHA-FA). A basal diet (BD) was formulated to contain 2.5 g methionine (Met) per kilogram of diet. The experimental diets included: (1) BD, (2) BD + 1 g DLM/kg, (3) BD + 2 g DLM/kg, (4) BD + 1.13 g MHA-FA/kg, (5) BD + 1.52 g MHA-FA/kg, (6) BD + 2.25 g MHA-FA/kg and (7) BD + 3.05 g MHA-FA/kg. Sixty weaned mixed-sex pigs were allotted to 5 treatment groups with 12 pig replicates per treatment in a randomized complete block design. During a 35-day experimental period, pigs in treatment group 1 received the BD whereas pigs in the other 4 treatment groups were allowed to choose between a pair of diets with either added 1 g DLM/kg in combination with either 1.13 or 1.52 g MHA-FA/kg or 2 g DLM/kg in combination with 2.25 or 3.05 g MHA-FA/kg, respectively. Pigs were housed in individual pens and had free access to feed and water. Daily feed intake (FI) was used as an indicator of diet preference. Cumulatively, pigs showed a preference (% of total FI) for the diet added with 1 g/kg DLM at 74% (P<0.05) in group 2, and 65% in group 3. Irrespective of the level of MHA-FA supplementation (2.25 or 3.05 g/kg), a preference for the diet supplemented with 2 g DLM/kg was 84% (P<0.05) in groups 4 and 5. During the entire period, pigs consistently (P<0.05) consumed more and preferred the diets supplemented with DLM more than the diets supplemented with MHA-FA in groups 2, 4 and 5. The preference for diets supplemented with DLM was more pronounced at higher Met supplementation levels. Feeding pigs a pair of diets supplemented with DLM or MHA-FA improved (P<0.05) the final body weight, daily weight gain, and FCR. The performance of pigs among the Met-supplemented groups was not different. In conclusion, when given a choice pigs preferred the diets supplemented with DLM more than the diets supplemented with liquid MHA-FA.     

Insulin-induced dissociation of its receptor into subunits: possible molecular concomitant of negative cooperativity

The detergent-solubilized avian insulin receptor retains negative cooperativity and other binding properties of the membrane bound form. On gel filtration the receptor elutes as a single peak with a Stokes radius of 72 A. Preincubation of the receptor with low levels of insulin leads to the formation of a second, smaller form with a Stokes radius of 40 A. The percent of receptor in this second peak is proportional to the insulin concentration and correlates well with the insulin-induced increase in dissociation rate (negative cooperativity). Both the isolated high molecular weight and the isolated low-molecular-weight forms of the receptor re-equilibrate in the presence of insulin and, upon refiltration of either isolated peak, both forms of the receptor are obtained. These results are compatible with a model of the insulin receptor in which a tetrameric form can dissociate to a monomeric form as a concomitant of negative cooperativity.     

TGFβ signaling in Tribolium: vertebrate-like components in a beetle

The cytokines of the TGFβ superfamily are highly conserved in evolution and elicit a diverse range of cellular responses in all metazoa. In Drosophila, the signaling pathways of the two TGFβ subfamilies, Activins and Bone Morphogenetic Proteins (BMPs), have been well studied. To address the question of whether the findings from Drosophila are representative of insects in general, we analyzed the components of TGFβ-signaling present in the genome of the beetle Tribolium castaneum. We were able to identify orthologs of the BMPs Decapentaplegic and Glass bottom boat, of the Activins Activinβ and Dawdle, as well as orthologs of the less well-known ligands Myoglianin and Maverick, together with orthologs of all TGFβ receptors and cytoplasmic signal transducers present in Drosophila. This indicates that the diversity of TGFβ signaling components is generally well conserved between Drosophila and Tribolium. However, the genome of the beetle—and of the bee Apis mellifera—lacks an ortholog of the Drosophila BMP Screw but does contain a vertebrate-like BMP10 homolog which is not found in Drosophila. Concerning BMP inhibitors, Tribolium displays an even more vertebrate-like ensemble of components. We found two orthologs of the vertebrate DAN family, Dan and Gremlin, and show embryonic expression of a vertebrate-like BAMBI ortholog, all of which are absent in Drosophila. This suggests that Tribolium might have retained a more ancestral composition of TGFβ signaling components and that TGFβ signaling underwent considerable change in the Drosophila lineage. Tribolium is an excellent model to study the function of these ancestral signaling components in insects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mating systems and predictors of relative reproductive success in a Cutthroat Trout subspecies of conservation concern

Mating systems and patterns of reproductive success in fishes play an important role in ecology and evolution. While information on the reproductive ecology of many anadromous salmonids (Oncorhynchus spp.) is well detailed, there is less information for nonanadromous species including the Yellowstone Cutthroat Trout (O. clarkii bouvieri), a subspecies of recreational angling importance and conservation concern. Using data from a parentage‐based tagging study, we described the genetic mating system of a migratory population of Yellowstone Cutthroat Trout, tested for evidence of sexual selection, and identified predictors of mating and reproductive success. The standardized variance in mating success (i.e., opportunity for sexual selection) was significantly greater for males relative to females, and while the relationship between mating success and reproductive success (i.e., Bateman gradient) was significantly positive for both sexes, a greater proportion of reproductive success was explained by mating success for males (r ² = 0.80) than females (r ² = 0.59). Overall, the population displayed a polygynandrous mating system, whereby both sexes experienced variation in mating success due to multiple mating, and sexual selection was variable across sexes. Tests for evidence of sexual selection indicated the interaction between mating success and total length best‐predicted relative reproductive success. We failed to detect a signal of inbreeding avoidance among breeding adults, but the group of parents that produced progeny were on average slightly less related than adults that did not produce progeny. Lastly, we estimated the effective number of breeders (N _b) and effective population size (N _e) and identified while N _b was lower than N _e, both are sufficiently high to suggest Yellowstone Cutthroat Trout in Burns Creek represent a genetically stable and diverse population.     

The importance of trimming reactions on asparagine-linked oligosaccharides for protein quality control

Although the biochemistry of early trimming reactions by glucosidases and ER mannosidases occurring on asparagine-linked oligosaccharides has been known for a long time, their involvement in quality control of protein folding has become apparent only more recently. Here we review the evidence for the involvement of specific oligosaccharide trimming intermediates such as Glc(1)Man(9)GlcNAc(2) and Man(8)GlcNAc(2) B isomer in this fundamental cellular process and the subcellular distribution of components of the protein quality control machinery which indicates the involvement of both the ER and pre-Golgi intermediates in this process. In addition, recent studies on the subcellular distribution of endomannosidase in conjunction with previously obtained biochemical data will be reviewed which demonstrate that an alternative deglucosylation pathway exists in pre-Golgi intermediates and the Golgi apparatus.     

Identification and characterization of DEDD2, a death effector domain-containing protein.

A novel Death Effector Domain-containing protein was identified, DEDD2, which is closest in amino acid sequence homology to death effector domain-containing DNA-binding protein, DEDD. DEDD2 mRNA is expressed widely in adult human tissues with highest levels in liver, kidney, and peripheral blood leukocytes. DEDD2 interacts with FLIP, but not with Fas-associated death domain (FADD) or caspase-8. Overexpression of DEDD2 induces moderate apoptosis and results in substantial sensitization to apoptosis induced by Fas (CD95/APO-1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L), or FADD. In contrast, Bax- or staurosporine-mediated cell death is not affected by expression of DEDD2. Fluorescence microscopy showed that overexpressed DEDD2 translocates to the nucleus, which is dependent on the presence of a bipartite nuclear localization signal in the DEDD2 protein. Mutagenesis studies revealed that the translocation of the DED of DEDD2 to the nucleus is essential for its pro-apoptotic activity. These findings suggest that DEDD2 is involved in the regulation of nuclear events mediated by the extrinsic apoptosis pathway.