Rhodopsin kinase is a key component in the shutdown of visual transduction. The phosphorylation of rhodopsin's C-terminus was evaluated using synthetic peptides derived from the last 12 amino acids (337-348) as substrates and their phosphorylated counterparts as inhibitors. It was found that synthetic peptides were phosphorylated at the serine residue corresponding to Ser-343 in the primary sequence of bovine rhodopsin. The phosphopeptides were prepared by incorporating into the peptide chain a trityl-protected serine derivative at the site destined to contain the phosphoryl group. The trityl group was selectively released with 20% (v/v) dichloroacetic acid; the free hydroxyl group was then phosphitylated with di-tert-butyl N,N-diethylphosphoramidite, and the resulting phosphite derivative was oxidized with m-chloroperoxybenzoic acid. The phosphopeptides were found to have a greater affinity for the kinase compared with their nonphosphorylated counterparts; for the peptides corresponding to residues 337-348 of rhodopsin the affinity increased in the order VSKTETSQVAPA < VSKTETS[PO3H2]QVAPA < VS[PO3H2]KTETS[PO3H2]QVAPA. The results are interpreted to support the cooperativity hypotheses proposed previously [Wilden, U., & Kühn, H. (1982) Biochemistry 21, 3014-3022; Aton, B. R., Litman, B. J., & Jackson, M. L. (1984) Biochemistry 23, 1737-1741].

Peptides of 10-12 amino acids in length, which overlapped with the sequence of the last 20 amino acids in the C-terminal tail of rhodopsin, were synthesized and used as substrates for rhodopsin kinase. In all cases the phosphorylation of the peptides was found to be greatly stimulated (> 20-fold) by the presence of light-activated rhodopsin (Rho*). The incorporation of 32P at seven Ser/Thr residues that are the potential sites of phosphorylation was quantified, and the results were analyzed in terms of two parameters. First, a global comparison of phosphorylation at each site was made when the propensity for the modification was found to be in the order: Ser 343 > Ser 338 > Thr 336 > Ser 334, Thr 342 > Thr 335, Thr 340. Second, the peptides were aligned on a hypothetical template with the residue to be phosphorylated occupying the P-position, and the manner in which the nature of the surrounding residues effected the phosphorylation was assessed. It was found that the optimal phosphorylation of the P-site Ser/Thr occurs if it has at least one residue on the amino side and five on the acyl side and also contains a neutral residue, preferably small (A, P, S, T) at the P+4 position.(ABSTRACT TRUNCATED AT 250 WORDS)

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit.

Skeletal muscle is inept in regenerating after traumatic injuries such as volumetric muscle loss (VML) due to significant loss of various cellular and acellular components. Currently, there are no approved therapies for the treatment of muscle tissue following trauma. In this study, biomimetic sponges composed of gelatin, collagen, laminin‐111, and FK‐506 were used for the treatment of VML in a rodent model. We observed that biomimetic sponge treatment improved muscle structure and function while modulating inflammation and limiting the extent of fibrotic tissue deposition. Specifically, sponge treatment increased the total number of myofibers, type 2B fiber cross‐sectional area, myosin: collagen ratio, myofibers with central nuclei, and peak isometric torque compared to untreated VML injured muscles. As an acellular scaffold, biomimetic sponges may provide a promising clinical therapy for VML.

Myeloid‐derived suppressor cells (MDSCs) are a population of immature myeloid immune cells that promote tumor progression and metastasis by suppressing both innate and adaptive immune responses. Exercise modulates immune function and is generally thought to protect against tumor growth and metastasis, yet the mechanisms behind this protection are largely unknown.PURPOSETo examine how exercise impacts circulating and tissue resident lymphocyte and MDSC populations.METHODSFemale mice were randomly assigned to treatment groups: exercise tumor (EX+TUM), sedentary tumor (SED+TUM), exercise (EX) or sedentary control (SED). EX groups underwent wheel running preconditioning for 6 weeks before tumor challenge. Following the 6‐week training period, animals in TUM groups were inoculated in the mammary fat pad with 1×104 4T1 mammary carcinoma cells and ran an additional 4‐weeks. Proportions of CD4+, CD8+, and CD335+ lymphocytes, as well as total MDSCs (CD11b+Ly6C+ Monocytic MDSCs (M‐MDSC) plus CD11b+Ly6G+ polymorphonuclear MDSCs (PMN‐MDSC)) were analyzed by flow cytometry in spleen, blood, and tumor.RESULTSNo effect of exercise was observed in CD4+ or CD8+ lymphocyte populations. The 4T1 mammary tumors significantly expanded MDSC populations in both spleen and blood regardless of exercise treatment. This tumor‐induced MDSC expansion was associated with enlarged spleens in sedentary animals (SED+TUM: 0.62g ± 0.11g) whereas the spleens in exercised animals with tumors were smaller (EX+TUM: 0.44g ± 0.11g) and did not differ from controls (EX: 0.08g ± 0.01g and SED: 0.11g ± 0.01g). Although there was no change in tumor volumes or tumor weights, exercise significantly reduced proportions of tumor‐infiltrating MDSCs (EX+TUM: 4.1% ± 1.6% vs. SED+TUM: 15.9% ± 2.3%). Further, tumor‐bearing exercised animals maintained a higher proportion of CD335+ natural killer cells in their spleens (EX+TUM: 0.6% ± 0.3%) relative to sedentary animals (SED+TUM: 0.4% ± 0.1%), which showed significant splenic CD335+ depletion relative to EX (1.3% ± 0.6%) controls.CONCLUSIONSThe observed reduction in tumor‐resident MDSCs suggests that exercise may improve antitumor immunity by decreasing MDSC‐related immune suppression in the tumor microenvironment and by preserving CD335+ lymphocyte populations. Exercise may reduce the severity of the aberrant myelopoiesis in tumor bearing animals as is evident by reduced splenomegaly in exercised animals.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.