Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation. Cx50D47A does not traffic properly, and homozygous mutant lenses show increased levels of the stress-responsive ␣B-crystallins. Therefore, we assessed whether expression of Cx50D47A led to endoplasmic reticulum (ER) stress in the lens in vivo. Although pharmacologic induction of ER stress can be transduced by three different pathways, we found no evidence for activation of the IRE1␣ or ATF6 pathways in Cx50D47A-expressing lenses. In contrast, heterozygous and homozygous Cx50D47A lenses showed an increase in phosphorylated PERK immunoreactivity and in the ratio of phosphorylated to total EIF2␣ (2.4-and 3.3-fold, respectively) compared with wild type. Levels of ATF4 were similar in wild type and heterozygous lenses but elevated in homozygotes (391%). In both heterozygotes and homozygotes, levels of calreticulin protein were increased (184 and 262%, respectively), as was Chop mRNA (1.9-and 12.4-fold, respectively). CHOP protein was increased in homozygotes (384%). TUNEL staining was increased in Cx50D47A lenses, especially in homozygous mice. Levels of two factors that may be pro-survival, Irs2 and Trib3, were greatly increased in homozygous lenses. These results suggest that expression of Cx50D47A induces ER stress, triggering activation of the PERK-ATF4 pathway, which potentially contributes to the lens pathology and leads to increased expression of anti-apoptotic factors, allowing cell survival.Proteins that do not traffic properly or are misfolded can cause endoplasmic reticulum (ER) 2 stress. To compensate for the disturbance of proteostasis, cells transduce ER stress through at least three different signaling pathways: (a) the inositol-requiring enzyme 1 (IRE1␣)-dependent, (b) the activating transcription factor 6 (ATF6)-dependent, and (c) the PRKR-like ER kinase (PERK)-dependent pathways. Prolonged and uncompensated ER stress triggers the unfolded protein response (UPR), which may ultimately target the cells for apoptosis.Many disease-linked connexin mutants show impaired cellular trafficking. One such mutant has a missense mutation in the lens gap junction protein, connexin50 (Cx50), affecting amino acid residue 47 (Cx50D47N in humans and Cx50D47A in mice) and is linked to congenital cataracts (1, 2). When expressed in transfected cells, Cx50D47N and Cx50D47A localize mostly within the ER (1). Lenses from mice expressing Cx50D47A have nuclear cataracts, reduced sizes, and impaired denucleation (3). Some immunoreactive Cx50 localizes intracellularly in lens epithelial cells from these mice (3). Homozygous Cx50D47A lenses have increased levels of ␣B-crystallins (3), which are responsive to several types of stress in different cell types, including lens epithelial cells (4 -7). These observations suggest that expression of a Cx50D47 mutant may cause ER stress in the lens and contribute to pathology in this organ.The current understanding of cellular responses to ER stress is based on many studies in cultured cells...

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Intrinsic healing of severed tendons shows a delay in a gain in breaking strength and the tendon becomes translucent. The cause of tendon translucence was investigated in suture-repaired rat Achilles tendon. The repair site with adjacent translucent tendon were evaluated histologically on day 10 by immunofluorescence and transmission electron microscopy. The healing tendon translucent region by hematoxylin-eosin staining had few inflammatory cells, polarized light birefringence showed thinner collagen fibers, immunofluorescence showed few myofibroblasts, and transmission electron microscopy revealed frayed, irregular thin collagen fibers. During embryogenesis, tendon fibers grow by the addition of discreet collagen fibril segment structures. The speculation is that collagen fibril segment structures are released from collagen fibers within the translucent tendon region for reuse during the regeneration of tendon collagen fibers during intrinsic tendon repair. Healing tendon translucence is related to a decrease in the diameter of collagen fibers by the release of collagen fibril segments within tendon bundles/fascicles.

A label-free mass spectrometric strategy was used to examine the effect of 5-fluorouracil (5-FU) on the primary and metastatic colon carcinoma cell lines, SW480 and SW620, with and without treatment. 5-FU is the most common chemotherapeutic treatment for colon cancer. Pooled biological replicates were analyzed by nanoLC-MS/MS and protein quantification was determined via spectral counting. Phenotypic and proteomic changes were evident and often similar in both cell lines. The SW620 cells were more resistant to 5-FU treatment, with an IC50 2.7-fold higher than that for SW480. In addition, both cell lines showed pronounced abundance changes in pathways relating to antioxidative stress response and cell adhesion remodeling due to 5-FU treatment. For example, the detoxification enzyme NQO1 was increasedwith treatment in both cell lines, while disparate members of the peroxiredoxin family, PRDX2 or PRDX5 and PRDX6, were elevated with 5-FU exposure in either SW480 or SW620, respectively. Cell adhesion associated proteins CTNNB1 and RhoA showed decreased expression with 5-FU treatment in both cell lines. The differential quantitative response in the proteomes of these patient-matched cell lines to drug treatment underscores the subtle molecular differences separating primary and metastatic cancer cells.

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