Effects of vitamin D metabolites on axolotl limb regeneration

Vitamin D is essential for normal metabolism of phosphorus and calcium, and differentiation of skeletal elements. 1,25 dihydroxyvitamin-D₃, the biologically active metabolite, acts as an induction/proliferation switch in various cell types and promotes chondrogenesis of chick limb bud mesenchymal cells. The function of vitamin D is mediated through its nuclear receptor, the vitamin D receptor (VDR). The proliferative actions of 1,25(OH)₂-D₃ on limb bud mesenchymal cells are similar to the ones produced by retinoids, such as all- trans retinoic acid (RA) or 9- cis retinoic acid (9- cis ). The retinoids have been shown to be compounds of extreme importance in the field of limb development and regeneration. In order to examine possible roles of vitamin D metabolites on limb regeneration, the effects of 1,25(OH)₂-D₃, 24,25(OH)₂-D₃ and KH1060 (a more potent metabolite) alone or in conjunction with all- trans RA or 9- cis RA on the regenerating axolotl limb. Vitamin D affects limb morphogenesis by generating abnormalities in skeletal elements. Synergism of vitamin D with retinoic acid in affecting pattern formation is suggested by the results.     

Prognostic impact of epigenetic classification in chronic lymphocytic leukemia: The case of subset #2

Based on the methylation status of 5 single CpG sites, a novel epigenetic classification of chronic lymphocytic leukemia (CLL) was recently proposed, classifying CLL patients into 3 clinico-biological subgroups with different outcome, termed memory like CLL (m-CLL), naïve like CLL (n-CLL), and a third intermediate CLL subgroup (i-CLL). While m-CLL and n-CLL patients at large corresponded to patients carrying mutated and unmutated IGHV genes, respectively, limited information exists regarding the less defined i-CLL group. Using pyrosequencing, we investigated the prognostic impact of the proposed 5 CpG signature in a well-characterized CLL cohort (135 cases), including IGHV-mutated and unmutated patients as well as clinically aggressive stereotyped subset #2 patients. Overall, we confirmed the signature's association with established prognostic markers. Moreover, in the presence of the IGHV mutational status, the epigenetic signature remained independently associated with both time-to-first-treatment and overall survival in multivariate analyses. As a prime finding, we observed that subset #2 patients were predominantly classified as i-CLL, probably reflecting their borderline IGHV mutational status (97–99% germline identity), though having a similarly poor prognosis as n-CLL patients. In summary, we validated the epigenetic classifier as an independent factor in CLL prognostication and provide further evidence that subset #2 is a member of the i-CLL group, hence supporting the existence of a third, intermediate epigenetic subgroup.     

The Combination of X-Ray Crystallography and Cryo-Electron Microscopy Provides Insight into the Overall Architecture of the Dodecameric Rvb1/Rvb2 Complex

The Rvb1/Rvb2 complex is an essential component of many cellular pathways. The Rvb1/Rvb2 complex forms a dodecameric assembly where six copies of each subunit form two heterohexameric rings. However, due to conformational variability, the way the two rings pack together is still not fully understood. Here, we present the crystal structure and two cryo-electron microscopy reconstructions of the dodecameric, full-length Rvb1/Rvb2 complex, all showing that the interaction between the two heterohexameric rings is mediated through the Rvb1/Rvb2-specific domain II. Two conformations of the Rvb1/Rvb2 dodecamer are present in solution: a stretched conformation also present in the crystal, and a compact conformation. Novel asymmetric features observed in the reconstruction of the compact conformation provide additional insight into the plasticity of the Rvb1/Rvb2 complex.     

Effect of Tetrahydrobiopterin on Serotonin Synthesis, Release, and Metabolism in Superfused Hippocampal Slices

The effects of 6R-5,6,7,8-tetrahydro-L-biopterin (6R-BH4), the in vivo cofactor for tryptophan hydroxylase, on the synthesis, release, and metabolism of serotonin were studied in superfused slices from rat hippocampus. 6R-BH4 did not alter the spontaneous release of [3H]serotonin but it did significantly increase release when slices were depolarized with 30 mM KCl. Under the same incubation conditions, 6R-BH4 altered neither the synthesis (basal or tryptophan-stimulated) nor the metabolism of serotonin in hippocampal slices. The synthetic pteridine 6-methyl-5,6,7,8-tetrahydropterin also augmented release under depolarizing conditions whereas biopterin, the oxidized form of 6R-BH4, did not. The 6S isomer of BH4, which is relatively inactive as a cofactor for tryptophan hydroxylase, was equipotent with 6R-BH4 in stimulating serotonin release. 6R-BH4 did not inhibit serotonin uptake nor did it function as a serotonin autoreceptor antagonist to increase release. A direct serotonin releasing effect of 6R-BH4, like that produced by p-chloroamphetamine, could also be ruled out. At suboptimal concentrations of extracellular calcium, the KCl-induced release of 3H was significantly reduced, yet the increase in release caused by BH4 remained the same in magnitude. It is concluded that 6R-BH4 increases the depolarization-induced release of serotonin through an interaction with the release mechanism itself, possibly by enhancing calcium influx or by increasing the sensitivity of the release mechanism to calcium. The effects of 6R-BH4 on serotonin release are independent from its function as the cofactor for tryptophan hydroxylase.     

5′ Phosphorylation of DNA in mammalian cells: Identification of a polymin P-precipitable polynucleotide kinase

Proteins that catayze 5′ phosphorylation of an oligodeozyribonucleotide substrate can be fractionated by polumin P treatment of whole cell extrats of calf thymus glands. Anion exchange chromatography on Q-Sepharose revealed three separable peaks of activity in the polymin P supernatant fraction, and one peak of activity in the Polymin P pellet fraction. The latter activity, polymin P-precipitable polynucleotide kinase (PP-PNK), was futher purified with a 1,500-fold increase of specific activity compared to the crude polymin fraction. Oligonucleotides, a dephosphorylated 2.9-kb EcoRI fragment, and poly(A) were phosphorylated by the enzyme preparation, but thymidine 3′monophosphate was not a substrate. PP-PNk preparations exhibited an apparent K_M of 52 μM for ATP and 8 μM for oligo dT₂₅. The enzyme preparation displayed no detectable 3′ phosphatase or cyclic 2′,3′ phosphohydrolase activities. The sedimentation coefficient of the PP-Pnk activity was 3.85 as determined by sucrose density gradient analysis; the stokes radius was 45 Å, leading to an estimated molecular mass of 72 kDa. The enzyme had a pH optimun in the neutral to alkaline range in several buffer systems and is distinct from the DNA Kinase with an acidic pH optimum previously described in calf thymus. © Wiley-Liss, Inc.     

Bridging knowledge gaps on the long road to regeneration: classical models meet stem cell manipulation and bioengineering

Research into tissue repair and regeneration is fought on many fronts. This article emphasizes that research into therapeutic tissue regeneration depends on diverse concepts and approaches. The interface between classical animal models, such as invertebrates and newts, and new opportunities, such as those afforded by mammalian stem cells, is in many respects unexpected. But this interface may make a reality of medicine's long-held dreams for tissue regeneration and organ replacement.