Human Fibroblast Sheet Promotes Human Pancreatic Islet Survival and Function In Vitro

In previous work, we engineered functional cell sheets using bone marrow-derived mesenchymal stem cells (BM-MSCs) to promote islet graft survival. In the present study, we hypothesized that a cell sheet using dermal fibroblasts could be an alternative to MSCs, and then we aimed to evaluate the effects of this cell sheet on the functional viability of human islets. Fibroblast sheets were fabricated using temperature-responsive culture dishes. Human islets were seeded onto fibroblast sheets. The efficacy of the fibroblast sheets was evaluated by dividing islets into three groups: the islets-alone group, the coculture with fibroblasts group, and the islet culture on fibroblast sheet group. The ultrastructure of the islets cultured on each fibroblast sheet was examined by electron microscopy. The fibroblast sheet expression of fibronectin (as a component of the extracellular matrix) was quantified by Western blotting. After 3 days of culture, islet viabilities were 70.2 ± 9.8%, 87.4 ± 5.8%, and 88.6 ± 4.5%, and survival rates were 60.3 ± 6.8%, 65.3 ± 3.0%, and 75.8 ± 5.6%, respectively. Insulin secretions in response to high-glucose stimulation were 5.1 ± 1.6, 9.4 ± 3.8, and 23.5 ± 12.4 μIU/islet, and interleukin-6 (IL-6) secretions were 3.0 ± 0.7, 5.1 ± 1.2, and 7.3 ± 1.0 ng/day, respectively. Islets were found to incorporate into the fibroblast sheets while maintaining a three-dimensional structure and well-preserved extracellular matrix. The fibroblast sheets exhibited a higher expression of fibronectin compared to fibroblasts alone. In conclusion, human dermal fibroblast sheets fabricated by tissue-engineering techniques could provide an optimal substrate for human islets, as a source of cytokines and extracellular matrix

Rapidly labelled ribonucleoprotein particles in rat liver cytoplasm and their relevance to the transport of messenger RNA

The appearance of rapidly labelled particles in the cytoplasm of rat liver has been followed in animals treated with 0.5 mg/kg actinomycin D to inhibit the synthesis of ribosomal subunits. Particles labelled under these conditions show a polydisperse sedimentation pattern on sucrose gradients, extending from 10 to 80 S with a maximum at 30 S. On CsCl gradients their density is 1.54 g/cm3. A component with a density of 1.38 g/cm3 has been observed under certain conditions, but this is considered to be an artifact. The RNA found in these particles has a polydisperse distribution on sucrose gradients extending from 7 to 35 S with a maximum at 17 S. Rapidly labelled non-ribosomal RNA is transported to the cytoplasm and becomes associated with polysomes in the presence of both actinomycin D and cycloheximide during short labelling periods. These results are discussed in relation to theories of the transport of mRNA from the nucleus to the cytoplasm

Changes in oxidative phosphorylation caused by ethyl βββ-trichloropropionate

The effects of ethyl βββ-trichloropropionate on respiration, ATPase activity, and the ATP/phosphate exchange reactions of rat liver mitochondria have been investigated. The major effect of the drug on respiration is a marked inhibition of ADP- or dinitrophenol-stimulated respiration with glutamate plus malate, succinate, or ascorbate plus tetramethyl-p-phenylene diamine as substrate. The inhibition is most marked with glutamate plus malate. Ethyl βββ-trichloropropionate at low concentration (up to 0.8 mm) also causes a slight stimulation of respiration in the absence of ADP or DNP when glutamate plus malate or succinate is the substrate. At similar concentrations to those at which it inhibits respiration, ethyl βββ-trichloropropionate stimulated mitochondrial ATPase activity to a very large extent. The relation between ATPase activity and drug concentration was sigmoid and the greatest rate obtained was higher than the maximum rate obtained with dinitrophenol. Dinitrophenol was antagonistic to ethyl βββ-trichloropropionate. Ethyl βββ-trichloropropionate inhibited the ATP/phosphate exchange reaction. The activities of a number of chloroalkyl esters related to ethyl βββ-trichloropropionate are compared and the connection between the results in the cell-free system and the biological effects of the compound in the whole animal are discussed