Unifying principles of regeneration I: Epimorphosis versus morphallaxis

Because research on regeneration has a long history, some classic definitions and concepts about regeneration which were established in earlier times have been retained without reconsideration for a long time, even though many relevant new findings have accumulated. To clarify the points on which research should be focused on for elucidating the mechanisms of regeneration, we should reconsider such classical definitions and principles of regeneration at the cellular and molecular level. Here, we consider two differing principles of regeneration which have been classically defined as 'epimorphosis' and 'morphallaxis', and propose the abandonment of these classical categories and their replacement by a new unifying principle in order to facilitate regeneration studies.     

Spontaneous atrial fibrillation initiated by triggered activity near the pulmonary veins in aged rats subjected to glycolytic inhibition

Aging and glycolytic inhibition (GI) are known to alter intracellular calcium ion (Ca(i)(2+)) handling in cardiac myocytes, causing early afterpotentials (EADs) and delayed afterpotentials. We hypothesized that aging and GI interact synergistically in intact hearts to generate EADs and triggered activity leading to atrial fibrillation (AF). We studied isolated and Langendorff-perfused hearts of young (age 3-5 mo, N = 8) and old (age 27-29 mo, N = 14) rats subjected to GI (0 glucose + 10 mmol/l pyruvate). Epicardial atrial activation maps were constructed using optical action potentials, while simultaneously monitoring Ca(i)(2+) by means of dual-voltage and calcium-sensitive fluorescent dyes. During GI, spontaneous AF occurred in 13 of 14 old but in no young rats. AF was initiated by EAD-induced triggered activity at the left atrial pulmonary vein junction (LA-PVJ). The triggered activity initially propagated as single wave front, but within 1 s degenerated into multiple wavelets. The EADs and triggered activity in the old atria were associated with significantly elevated diastolic Ca(i)(2+) levels at the LA-PVJ, where the time constant tau of the Ca(i)(2+) transient decline and action potential duration were significantly (P < 0.01) prolonged compared with atrial sites 5 mm away from LA-PVJ. During GI and rapid atrial pacing, spatially discordant APD and Ca(i)(2+) transient alternans developed in the old but not young atria, leading to AF. Atria in old rats had significantly more fibrotic tissue than atria in young rats. We conclude that GI interacts with the aged and fibrotic atria to amplify Ca(i)(2+) handling abnormalities that facilitate EAD-mediated triggered activity and AF.     

Melanogenesis inhibition by an oolong tea extract in b16 mouse melanoma cells and UV-induced skin pigmentation in brownish guinea pigs

To investigate the new physiological functions of oolong tea, the effects on melanogenesis were studied. An oolong tea extract inhibited melanogenesis without affecting cell growth in B16 mouse melanoma cells. However, the oolong tea extract hardly showed any inhibitory effect on mushroom tyrosinase in a cell-free system. The effects of an oolong tea extract on the intracellular tyrosinase level in B16 cells were therefore studied. All the levels of activity, protein and mRNA were decreased in the oolong tea extract-treated cells. We also investigated the inhibitory effects of oolong tea on the pigmentation induced by ultraviolet B (UVB) by using brownish guinea pigs in vivo. The number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes increased by UVB was repressed by an oral administration of oolong tea. These results imply that oolong tea might be effective in whitening and that its inhibitory effect on melanogenesis was involved in the decrease of intracellular tyrosinase at the mRNA level.     

Screening of carbon dioxide-requiring extreme oligotrophs from soil

We screened soil samples for CO(2)-requiring extreme oligotrophs similar to Rhodococcus erythropolis N9T-4, which can grow on a basal salt agar medium without an organic carbon source. From 387 soil samples, three isolates were obtained and identified as Streptomyces spp. by 16S rDNA analysis. The isolates required gaseous CO(2) for growth and grew on a basal salt medium solidified by silica gel. These results suggest that such CO(2)-requiring oligotrophs occur widely in nature.     

Tumor Necrosis Factor (TNF)-α-induced Repression of GKAP42 Protein Levels through cGMP-dependent Kinase (cGK)-Iα Causes Insulin Resistance in 3T3-L1 Adipocytes

Insulin receptor substrates (IRSs) have been shown to be major mediators of insulin signaling. Recently, we found that IRSs form high-molecular weight complexes, and here, we identify by yeast two-hybrid screening a novel IRS-1-associated protein: a 42-kDa cGMP-dependent protein kinase-anchoring protein (GKAP42). GKAP42 knockdown in 3T3-L1 adipocytes suppressed insulin-dependent IRS-1 tyrosine phosphorylation and downstream signaling, resulting in suppression of GLUT4 translocation to plasma membrane induced by insulin. In addition, GLUT4 translocation was also suppressed in cells overexpressing GKAP42-N (the IRS-1 binding region of GKAP42), which competed with GKAP42 for IRS-1, indicating that GKAP42 binding to IRS-1 is required for insulin-induced GLUT4 translocation. Long term treatment of 3T3-L1 adipocytes with TNF-α, which induced insulin resistance, significantly decreased the GKAP42 protein level. We then investigated the roles of cGMP-dependent kinase (cGK)-Iα, which bound to GKAP42, in these changes. cGK-Iα knockdown partially rescued TNF-α-induced decrease in GKAP42 and impairment of insulin signals. These data indicated that TNF-α-induced repression of GKAP42 via cGK-Iα caused reduction of insulin-induced IRS-1 tyrosine phosphorylation at least in part. The present study describes analysis of the novel TNF-α-induced pathway, cGK-Iα-GKAP42, which regulates insulin-dependent signals and GLUT4 translocation.     

A set of external reference controls/probes that enable quality assurance between different microarray platforms

RNA external standards, although important to ensure equivalence across many microarray platforms, have yet to be fully implemented in the research community. In this article, a set of unique RNA external standards (or RNA standards) and probe pairs that were added to total RNA in the samples before amplification and labeling are described. Concentration–response curves of RNA external standards were used across multiple commercial DNA microarray platforms and/or quantitative real-time polymerase chain reaction (RT–PCR) and next-generation sequencing to identify problematic assays and potential sources of variation in the analytical process. A variety of standards can be added in a range of concentrations spanning high and low abundances, thereby enabling the evaluation of assay performance across the expected range of concentrations found in a clinical sample. Using this approach, we show that we are able to confirm the dynamic range and the limit of detection for each DNA microarray platform, RT–PCR protocol, and next-generation sequencer. In addition, the combination of a series of standards and their probes was investigated on each platform, demonstrating that multiplatform calibration and validation is possible.     

Gefitinib-sensitive EGFR lacking residues 746-750 exhibits hypophosphorylation at tyrosine residue 1045, hypoubiquitination, and impaired endocytosis

Gefitinib-sensitive nonsmall cell lung cancers (NSCLC) are characterized by somatic mutations in the kinase domain of epidermal growth factor receptor (EGFR). The mutant EGFR forms are reported to mediate characteristic signal transduction pathways that are different from those mediated by the wild-type EGFR and are involved in transformation in vivo. We have examined signal transduction pathways initiated from a frequently identified gefitinib-sensitizing mutant EGFR lacking residues 746-750 by employing a mouse fibroblast cell line that is free of endogenous EGFR and transiently transfected COS-7 cells. Upon EGF stimulation, the deletion-mutant EGFR mediated prolonged downstream signals. The analysis of the phosphotyrosine patterns of the receptor revealed that the deletion-mutant EGFR lacked phosphorylation at tyrosine residue 1045, which is the major binding site of Cbl. The EGF-induced endocytosis of the deletion-mutant EGFR was impaired. The ubiquitination and downregulation of the deletion-mutant EGFR were also reduced. On the other hand, another mutant, EGFR, possessing a L858R substitution, exhibited phosphorylation at 1045 and its downstream signalings were not prolonged. These data suggest that the signal transduction pathways initiated from these mutant forms are different, and that impaired endocytosis might be responsible for the prolonged signals mediated by the deletion-mutant EGFR.