A positive regulatory role for suppressor of cytokine signaling 1 in IFN-gamma-induced MHC class II expression in fibroblasts

Suppressor of cytokine signaling 1 (SOCS1) is rapidly induced following stimulation by several cytokines. SOCS1 negatively regulates cytokine receptor signal transduction by inhibiting Janus family tyrosine kinases. Lack of such feedback regulation underlies the premature death of SOCS1(-/-) mice due to unbridled IFN-gamma signaling. We used mouse embryo fibroblasts derived from SOCS1(-/-) mice to investigate the role of SOCS1 in IFN-gamma signaling pathways. SOCS1(-/-) fibroblasts were exquisitely sensitive to the IFN-gamma-mediated growth arrest and showed sustained STAT1 phosphorylation. However, SOCS1(-/-) fibroblasts were inefficient in MHC class II surface expression following IFN-gamma stimulation, despite a marked induction of the MHC class II transactivator and MHC class II gene expression. Retroviral transduction of wild-type SOCS1 relieved the growth-inhibitory effects of IFN-gamma in SOCS1(-/-) fibroblasts by inhibiting STAT1 activation. SOCS1R105K, carrying a mutation within the phosphotyrosine-binding pocket of the Src homology 2 domain, did not inhibit STAT1 phosphorylation, yet considerably inhibited IFN-gamma-mediated growth arrest. Strikingly, expression of SOCS1R105K restored the IFN-gamma-induced MHC class II expression in SOCS1(-/-) cells, indicating that expression of SOCS1 facilitates MHC class II expression in fibroblasts. Our results show that SOCS1, in addition to its negative regulatory role of inhibiting Janus kinases, has an unanticipated positive regulatory function in retarding the degradation of IFN-gamma-induced MHC class II proteins in fibroblasts.     

Quantification of red myotomal muscle volume and geometry in the shortfin mako shark (Isurus oxyrinchus) and the salmon shark (Lamna ditropis) using T1-weighted magnetic resonance imaging

T1-weighted magnetic resonance imaging (MRI) in conjunction with image and segmentation analysis (i.e., the process of digitally partitioning tissues based on specified MR image characteristics) was evaluated as a noninvasive alternative for differentiating muscle fiber types and quantifying the amounts of slow, red aerobic muscle in the shortfin mako shark (Isurus oxyrinchus) and the salmon shark (Lamna ditropis). MRI-determinations of red muscle quantity and position made for the mid-body sections of three mako sharks (73.5-110 cm fork length, FL) are in close agreement (within the 95% confidence intervals) with data obtained for the same sections by the conventional dissection method involving serial cross-sectioning and volumetric analyses, and with previously reported findings for this species. The overall distribution of salmon shark red muscle as a function of body fork length was also found to be consistent with previously acquired serial dissection data for this species; however, MR imaging revealed an anterior shift in peak red muscle cross-sectional area corresponding to an increase in body mass. Moreover, MRI facilitated visualization of the intact and anatomically correct relationship of tendon linking the red muscle and the caudal peduncle. This study thus demonstrates that MRI is effective in acquiring high-resolution three-dimensional digital data with high contrast between different fish tissue types. Relative to serial dissection, MRI allows more precise quantification of the position, volume, and other details about the types of muscle within the fish myotome, while conserving specimen structural integrity.     

Molecular Determinants of Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Binding to Transient Receptor Potential V1 (TRPV1) Channels

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P₂ is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P₂ binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P₂ interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P₂ behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P₂ binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P₂ in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P₂ binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.     

Genetic Polymorphisms Associated with Hearing Threshold Shift in Subjects during First Encounter with Occupational Impulse Noise

Noise-induced hearing loss (NIHL) is the most significant occupational health issue worldwide. We conducted a genome-wide association study to identify single-nucleotide polymorphisms (SNPs) associated with hearing threshold shift in young males undergoing their first encounter with occupational impulse noise. We report a significant association of SNP rs7598759 (p < 5 x 10^-7; p = 0.01 after permutation and correction; Odds Ratio = 12.75) in the gene coding for nucleolin, a multifunctional phosphoprotein involved in the control of senescence and protection against apoptosis. Interestingly, nucleolin has been shown to mediate the anti-apoptotic effect of HSP70, a protein found to prevent ototoxicity and whose polymorphisms have been associated with susceptibility to NIHL. Increase in nucleolin expression has also been associated with the prevention of apoptosis in cells undergoing oxidative stress, a well-known metabolic sequela of noise exposure. To assess the potential role of nucleolin in hearing loss, we tested down-regulation of nucleolin in cochlear sensory cells HEI-OC1 under oxidative stress conditions and report increased sensitivity to cisplatin, a chemotherapeutic drug with ototoxic side effects. Additional SNPs were found with suggestive association (p < 5 x 10^-4), of which 7 SNPs were located in genes previously reported to be related to NIHL and 43 of them were observed in 36 other genes previously not reported to be associated with NIHL. Taken together, our GWAS data and in vitro studies reported herein suggest that nucleolin is a potential candidate associated with NIHL in this population.     

Use of chitin to improve a Beauveria bassiana alginate-pellet formulation

A study was undertaken to evaluate optimum concentrations of chitin in sodium alginate pellet formulations to enhance conidia production. Chitin concentrations tested were 0, 0.5, 1, 2, 3 and 4% (w/v), with (2%, w/v) or without wheat bran. The different chitin-wheat bran pellet combinations were prepared with Beauveria bassiana isolate Qu-B306 at 10⁸ conidia mL^-1. After 21 days of incubation in a humid chamber at 28°C, conidia production was assessed. Improvements up to three times the initial conidia number were achieved using 2% chitin and 2% wheat bran. Higher levels of chitin decreased the number of conidia per pellet. For all chitin concentrations, conidia number increased with the addition of wheat bran (P≤0.05). Contamination by saprophytic fungi was reduced by the incorporation of chitin in the pellet formulation.     

Evolution of high-performance swimming in sharks: transformations of the musculotendinous system from subcarangiform to thunniform swimmers

In contrast to all other sharks, lamnid sharks perform a specialized fast and continuous "thunniform" type of locomotion, more similar to that of tunas than to any other known shark or bony fish. Within sharks, it has evolved from a subcarangiform mode. Experimental data show that the two swimming modes in sharks differ remarkably in kinematic patterns as well as in muscle activation patterns, but the morphology of the underlying musculotendinous system (red muscles and myosepta) that drives continuous locomotion remains largely unknown. The goal of this study was to identify differences in the musculotendinous system of the two swimming types and to evaluate these differences in an evolutionary context. Three subcarangiform sharks (the velvet belly lantern shark, Etmopterus spinax, the smallspotted catshark, Scyliorhinus canicula, and the blackmouth catshark, Galeus melanostomus) from the two major clades (two galeans, one squalean) and one lamnid shark, the shortfin mako, Isurus oxyrhinchus, were compared with respect to 1) the 3D shape of myomeres and myosepta of different body positions; 2) the tendinous architecture (collagenous fiber pathways) of myosepta from different body positions; and 3) the association of red muscles with myoseptal tendons. Results show that the three subcarangiform sharks are morphologically similar but differ remarkably from the lamnid condition. Moreover, the "subcarangiform" morphology is similar to the condition known from teleostomes. Thus, major features of the "subcarangiform" condition in sharks have evolved early in gnathostome history: Myosepta have one main anterior-pointing cone and two posterior-pointing cones that project into the musculature. Within a single myoseptum cones are connected by longitudinally oriented tendons (the hypaxial and epaxial lateral and myorhabdoid tendons). Mediolaterally oriented tendons (epineural and epipleural tendons; mediolateral fibers) connect vertebral axis and skin. An individual lateral tendon spans only a short distance along the body (a fraction between 0.05 and 0.075 of total length, L, of the shark). This span is similar in all tendons along the body. Red muscles insert into the midregion of the lateral tendons. The shortfin mako differs substantially from this condition in several respects: Red muscles are internalized and separated from white muscles by a sheath of lubricative connective tissue. They insert into the anterior part of the hypaxial lateral tendon. Rostrocaudally, this tendon becomes very distinct and its span increases threefold (0.06L anteriorly to 0.19L posteriorly). Mediolateral fibers do not form distinct epineural/epipleural tendons in the mako. Since our morphological findings are in good accordance with experimental data it seems likely that the thunniform swimming mode has evolved along with the described morphological specializations.     

Evaluation of chitosans and Pichia guillermondii as growth inhibitors of Penicillium digitatum

Chitosans were obtained by room-temperature-homogeneous-deacetylation (RTHD) and freeze-pump-out-thaw-heterogeneous-deacetylation (FPT) from chitins purified from fermentations. Commercial chitosan was deacetylated by three-FPT-cycles. Chitosans and Pichia guillermondii were evaluated on the growth of Penicillium digitatum. Medium molecular weight (M(W)) chitosans displayed higher inhibitory activity against the yeast than low M(W) biopolymers. Chitosans with low degree of acetylation (DA) were inhibitory for yeast and mould. Therefore, a low M(W) and high DA chitosan was selected for use against moulds combined with yeasts. Biopolymer and yeasts presented an additive effect, since chitosans were effective to delay spore germination, whereas yeast decreased apical fungal growth.     

Computational modeling study of functional microdomains in cannabinoid receptor type 1

The seven transmembrane helices (TMH) G-protein-coupled receptors (GPCRs) constitute one of the largest superfamily of signaling proteins found in mammals. Some of its members, in which the cannabinoid (CB) receptors are included, stand out because their functional states can be modulated by a broad spectrum of effector molecules. The relative ligand promiscuity exhibited by these receptors could be related with particular attributes conferred by their molecular architecture and represents a motivating issue to be explored. In this regard, this study represents an effort to investigate the cannabinoid receptor type 1 (CB1) ligand recognition plasticity, using comparative modeling, molecular dynamics (MD) simulations and docking. Our results suggest that a cooperative set of subtle structural rearrangements within the TMHs provide to the CB1 protein the plasticity to reach alternate configurations. These changes include the relaxation of intramolecular constraints, the rotations, translations and kinks of the majority of TMHs and the reorganization of the ligand binding cavities.     

Imprinting of CCR9 on CD4 T cells requires IL-4 signaling on mesenteric lymph node dendritic cells

It has recently been shown that IL-4 can educate dendritic cells (DC) to differentially affect T cell effector activity. In this study, we show that IL-4 can also act upon DC to instruct naive T cells to express the gut-associated homing receptor CCR9. Thus, effector T cells generated after coculture with mesenteric lymph node (MLN)-DC show a higher expression of CCR9 when activated in the presence of IL-4. In contrast, IL-4 had no effect on CCR9 expression when naive T cells were polyclonally activated in the absence of MLN-DC, suggesting that the effect of IL-4 on CCR9 expression passed through DC. Indeed, T cells activated by MLN-DC from IL-4Ralpha(-/-) mice showed a much lower CCR9 expression and a greatly reduced migration to the small intestine than T cells activated by wild-type MLN-DC even in the presence of IL-4. Consistent with the finding that the vitamin A metabolite retinoic acid (RA) induces gut-homing molecules on T cells, we further demonstrate that IL-4 up-regulated retinaldehyde dehydrogenase 2 mRNA on MLN-DC, a critical enzyme involved in the synthesis of RA. Moreover, LE135, a RA receptor antagonist, blocked the increased expression of CCR9 driven by IL-4-treated MLN-DC. Thus, besides the direct effect of RA on T cell gut tropism, our results show that the induction of a gut-homing phenotype on CD4(+) T cells is also influenced by the effect of IL-4 on gut-associated DC.