Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13.

Although the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK- populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.

Methodological considerations for the calculation of cumulative compression exposure of the lumbar spine: a sensitivity analysis on joint model and time standardization approaches.

Cumulative lumbar spine loading has attracted much attention as a factor associated with the development of low back pain. While evidence supports cumulative loading to be a plausible mechanism in explaining several workplace injuries, research establishing a threshold limit value (TLV) for cumulative spine loading has been challenging. The lack of a TLV or even a trend towards harmful cumulative load values may suggest that methodological considerations are greatly influencing the results. This paper examines the impact of different joint models (single muscle equivalent, an electromyography-based third order polynomial, a modified version of the polynomial and a hybrid approach) to determine cumulative spine compression, as well as the importance of time standardization in the calculation of a daily cumulative loading dose. Findings demonstrated that the polynomial predicted cumulative compression values were 43-53% higher than those with all other models tested and the single muscle equivalent predicted loads 18% higher than loads predicted using a modified polynomial. Profound differences between modelling approaches suggest that caution should be taken when selecting a muscle model to determine cumulative spine compressive loading. Time standardized cumulative compression values were found to be 28.3% greater than non-standardized estimates, illustrating the importance of selecting a standard time frame in the calculation of cumulative spine compression.

Mutation of the ATP-binding pocket of SSA1 indicates that a functional interaction between Ssa1p and Ydj1p is required for post-translational translocation into the yeast endoplasmic reticulum.

The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.

Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation into the yeast ER.

The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST-63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST-63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST-63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.

Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum.

To determine whether mitochondrial hsp70 (mHsp70) could substitute for the endoplasmic retuculum (ER) Hsp70 (BiP) during protein translocation, we assembled ER-derived reconstituted proteoliposomes supplemented with either protein. We found that only BiP restored translocation in kar2 mutant vesicles and stimulated translocation approximately 3-fold in wild type proteoliposomes. mHsp70 associated poorly with both a BiP binding (DnaJ) domain of Sec63p and an ER precursor, and its ATPase activity was poorly enhanced upon incubation with the DnaJ domain. In contrast, BiP bound to the Sec63p-DnaJ domain in an ATP-dependent manner and its ATPase activity was stimulated significantly by this polypeptide. We conclude that mHsp70 is unable to support protein translocation into the ER because it fails to associate productively with Sec63p and a precursor.

The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain.

Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four- to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.