BAG-6 is essential for selective elimination of defective proteasomal substrates

BAG-6/Scythe/BAT3 is a ubiquitin-like protein that was originally reported to be the product of a novel gene located within the human major histocompatibility complex, although the mechanisms of its function remain largely obscure. Here, we demonstrate the involvement of BAG-6 in the degradation of a CL1 model defective protein substrate in mammalian cells. We show that BAG-6 is essential for not only model substrate degradation but also the ubiquitin-mediated metabolism of newly synthesized defective polypeptides. Furthermore, our in vivo and in vitro analysis shows that BAG-6 interacts physically with puromycin-labeled nascent chain polypeptides and regulates their proteasome-mediated degradation. Finally, we show that knockdown of BAG-6 results in the suppressed presentation of MHC class I on the cell surface, a procedure known to be affected by the efficiency of metabolism of defective ribosomal products. Therefore, we propose that BAG-6 is necessary for ubiquitin-mediated degradation of newly synthesized defective polypeptides.     

Regulation of Toll-like receptor 4 expression in mouse colon by macrophage migration inhibitory factor

Recent studies have indicated that macrophage migration inhibitory factor (MIF) and Toll-like receptor (TLR) play an important role in the regulation of innate immune responses. In this study, we investigated the effect of MIF on the expression of TLR4, a receptor that recognizes lipopolysaccharide, in colon using MIF-deficient mice. TLR4 mRNA expression in the colon tissues was determined by northern blot analysis. Western blot analysis and immunohistochemistry in the colon tissues were performed to evaluate the expression of TLR4 protein. The expressions of TLR4 mRNA and protein were remarkably down-regulated in colon tissues of MIF-deficient mice compared with wild-type mice and up-regulated by treatment with recombinant MIF. Immunohistochemical study revealed the presence of TLR4–positive staining in mononuclear cells in the lamina propria and intraepithelial mononuclear cells as well as weak staining in epithelial cells and crypts in colon tissues of wild-type mice. In contrast, MIF-deficient mice did not show TLR4-positive staining in the colonic mucosa. In MIF-deficient mice injected with recombinant mouse MIF (rMIF), TLR4-positive staining cells were observed in colon tissues similar to the findings in wild-type mice. Administration of dextran sulfate sodium (DSS) up-regulated the expression of TLR4 in the colons of WT mice but not in those of MIF-deficient mice. Furthermore, pretreatment with rMIF up-regulated the expression of TLR4 in response to DSS in MIF-deficient mice. Our results suggest that MIF affects the expression of TLR4 in mouse colon under both normal and colitic conditions.An erratum to this article can be found at     

Comparative potencies of botulinum toxin for infant and adult mice

Culture fluids of 4 type A, 6 type B, and 1 type EClostridium botulinum strains were compared for their toxicities in 9–11-day-old (infant) and adult mice. In intraperitoneal challenges, most toxins were about equally potent for infants and adults; the exceptions were 4 type B preparations, which were 500 to 5,000 times more lethal for infants. In orogastric challenges, samples were 10 to >15,000 times more toxic for infants, and the previously mentioned type B toxins constituted a group having the highest relative toxicity for infants.     

Comparative analysis of DNA alkylation by conjugates between pyrrole–imidazole hairpin polyamides and chlorambucil or seco-CBI

We investigated sequence-specific DNA alkylation using conjugates between the N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide and the DNA alkylating agent, chlorambucil, or 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). Polyamide–chlorambucil conjugates 1–4 differed in the position at which the DNA alkylating chlorambucil moiety was bound to the Py–Im polyamide. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) revealed that chlorambucil conjugates 1–4 alkylated DNA at the sequences recognized by the Py–Im polyamide core moiety. Reactivity and sequence specificity were greatly affected by the conjugation position, which reflects the geometry of the alkylating agent in the DNA minor groove. Polyamide–seco-CBI conjugate 5 was synthesized to compare the efficacy of chlorambucil with that of seco-CBI as an alkylating moiety for Py–Im polyamides. Denaturing PAGE analysis revealed that DNA alkylation activity of polyamide–seco-CBI conjugate 5 was similar to that of polyamide–chlorambucil conjugates 1 and 2. In contrast, the cytotoxicity of conjugate 5 was superior to that of conjugates 1–4. These results suggest that the seco-CBI conjugate was distinctly active in cells compared to the chlorambucil conjugates. These results may contribute to the development of more specific and active DNA alkylating agents.     

Germline recombination in a novel Cre transgenic line,Prl3b1‐Cre mouse

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1‐cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL‐2) protein in placenta along with increased expression toward the end of pregnancy. PL‐2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1‐cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1‐cre;R26GRR mice revealed that tdsRed‐positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1‐cre;R26GRR testes suggested that Cre‐mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1‐cre mice line provides a unique resource to understand testicular germ‐cell development. genesis 54:389–397, 2016. © 2016 Wiley Periodicals, Inc.     

Requirement of tryptophan hydroxylase during development for maturation of sensorimotor gating

Deficits in sensorimotor gating, a function to focus on the most salient stimulus, could lead to a breakdown of cognitive integrity, and could reflect the "flooding" by sensory overload and cognitive fragmentation seen in schizophrenia. Sensorimotor gating emerges at infancy, and matures during childhood. The mechanisms that underlie its development are largely unclear. Here, we screened the mouse genome, and found that tryptophan hydroxylase (TPH) is implicated in the maturation of sensorimotor gating. TPH, an enzyme involved in the biosynthesis of serotonin, proved to be required only during the weaning period for maturation of sensorimotor gating, but was dispensable for its emergence. Proper serotonin levels during development underlie the mature functional architecture for sensorimotor gating via appropriate actin polymerization. Thus, maintaining proper serotonin levels during childhood may be important for mature sensorimotor gating in adulthood.     

Estimation of luminal mucin content in rats by measurement of O-linked oligosaccharide chains and direct ELISA

Changes in the small intestinal mucin contents in rats were evaluated by two methods, viz., a newly established ELISA and a method based on the measurement of O-linked oligosaccharide chains (OSC) as a mucin marker. Significant correlation was observed between the values of ELISA-derived mucins and OSC. The results confirm the usefulness of measurement of OSC as an alternative method for mucin determination.     

Developmental changes in the regulation of calcium-dependent neurite outgrowth

Intracellular calcium ions (Ca²⁺) have an essential role in the regulation of neurite outgrowth, but how outgrowth is controlled remains largely unknown. In this study, we examined how the mechanisms of neurite outgrowth change during development in chick and mouse dorsal root ganglion neurons. 2APB, a potent inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R), inhibited neurite outgrowth at early developmental stages, but not at later stages. In contrast, pharmacological inhibition with Ni²⁺, Cd²⁺, or dantrolene revealed that ryanodine receptor (RyR)-mediated Ca²⁺-induced Ca²⁺ release (CICR) was involved in neurite outgrowth at later stage, but not at early stages. The distribution of IP₃R and RyR in growth cones also changed during development. Furthermore, pharmacological inhibition of the Ca²⁺-calmodulin-dependent phosphatase calcineurin with FK506 reduced neurite outgrowth only at early stages. These data suggest that the calcium signaling that regulates neurite outgrowth may change during development from an IP₃R-mediated pathway to a RyR-mediated pathway.     

Proteomic analysis of highly purified peroxisomes from etiolated soybean cotyledons

To identify previously unknown peroxisomal proteins, we establishedan optimized method for isolating highly purified peroxisomesfrom etiolated soybean cotyledons using Percoll density gradientcentrifugation followed by iodixanol density gradient centrifugation.Proteins in highly purified peroxisomes were separated by two-dimensionalPAGE. We performed peptide mass fingerprinting of proteins separatedin the gel with matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry and used the peptide mass fingerprintsto search a non-redundant soybean expressed sequence tag database.We succeeded in assigning 92 proteins to 70 sequences in thedatabase. Among them, proteins encoded by 30 sequences werejudged to be located in peroxisomes. These included enzymesfor fatty acid β-oxidation, the glyoxylate cycle, photorespiratoryglycolate metabolism, stress response and metabolite transport.We also show experimental evidence that plant peroxisomes containa short-chain dehydrogenase/reductase family protein, enoyl-CoAhydratase/isomerase family protein, 3-hydroxyacyl-CoA dehydrogenase-likeprotein and a voltage-dependent anion-selective channel protein.