Global regulation of alternative splicing during myogenic differentiation

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely to be important in biological transitions such as cellular differentiation, or response to environmental stimuli. To assess the extent and significance of AS in myogenesis, we used splicing-sensitive microarray analysis of differentiating C2C12 myoblasts. We identified 95 AS events that undergo robust splicing transitions during C2C12 differentiation. More than half of the splicing transitions are conserved during differentiation of avian myoblasts, suggesting the products and timing of transitions are functionally significant. The majority of splicing transitions during C2C12 differentiation fall into four temporal patterns and were dependent on the myogenic program, suggesting that they are integral components of myogenic differentiation. Computational analyses revealed enrichment of many sequence motifs within the upstream and downstream intronic regions near the alternatively spliced regions corresponding to binding sites of splicing regulators. Western analyses demonstrated that several splicing regulators undergo dynamic changes in nuclear abundance during differentiation. These findings show that within a developmental context, AS is a highly regulated and conserved process, suggesting a major role for AS regulation in myogenic differentiation.     

Structure-based optimization of 1H-imidazole-2-carboxamides as Axl kinase inhibitors utilizing a Mer mutant surrogate

Axl has been a target of interest in the oncology field for several years based on its role in various oncogenic processes. To date, no wild-type Axl crystal structure has been reported. Herein, we describe the structure-based optimization of a novel chemotype of Axl inhibitors, 1H-imidazole-2-carboxamide, using a mutated kinase homolog, Mer(I650M), as a crystallographic surrogate. Iterative optimization of the initial lead compound (1) led to compound (21), a selective and potent inhibitor of wild-type Axl. Compound (21) will serve as a useful compound for further in vivo studies.     

Overlapping motifs (PTAP and PPEY) within the Ebola virus VP40 protein function independently as late budding domains: involvement of host proteins TSG101 and VPS-4

The VP40 protein of Ebola virus can bud from mammalian cells in the form of lipid-bound, virus-like particles (VLPs), and late budding domains (L-domains) are conserved motifs (PTAP, PPxY, or YxxL; where "x" is any amino acid) that facilitate the budding of VP40-containing VLPs. VP40 is unique in that potential overlapping L-domains with the sequences PTAP and PPEY are present at amino acids 7 to 13 of VP40 (PTAPPEY). L-domains are thought to function by interacting with specific cellular proteins, such as the ubiquitin ligase Nedd4, and a component of the vacuolar protein sorting (vps) pathway, tsg101. Mutational analysis of the PTAPPEY sequence of VP40 was performed to understand further the contribution of each individual motif in promoting VP40 budding. In addition, the contribution of tsg101 and a second member of the vps pathway, vps4, in facilitating budding was addressed. Our results indicate that (i) both the PTAP and PPEY motifs contribute to efficient budding of VP40-containing VLPs; (ii) PTAP and PPEY can function as L-domains when separated and moved from the N terminus (amino acid position 7) to the C terminus (amino acid position 316) of full-length VP40; (iii) A VP40-PTAP/tsg101 interaction recruits tsg101 into budding VLPs; (iv) a VP40-PTAP/tsg101 interaction recruits VP40 into lipid raft microdomains; and (v) a dominant-negative mutant of vps4 (E228Q), but not wild-type vps4, significantly inhibited the budding of Ebola virus (Zaire). These results provide important insights into the complex interplay between viral and host proteins during the late stages of Ebola virus budding.     

Demographic,seed and microsite limitations to seedling recruitment in semi-arid mine site restoration

Plant and Soil - Understanding limitations to plant recruitment is a key element in devising effective restoration of semi-arid ecosystems: only when these limitations are identified can management...     

Activation of cardiac AMP-activated protein kinase by LKB1 expression or chemical hypoxia is blunted by increased Akt activity

AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac energy substrate utilization and can be negatively regulated by Akt activation in the heart. It has recently been shown that Akt directly phosphorylates AMPKalpha(1)/alpha(2) on Ser(485/491) in vitro and prevents the AMPK kinase (AMPKK) LKB1 from phosphorylating AMPKalpha at its primary activation site, Thr(172) (S Horman, D Vertommen, R Heath, D Neumann, V Mouton, A Woods, U Schlattner, T Wallimann, D Carling, L Hue, and MH Rider. J Biol Chem 281: 5335-5340, 2006). To determine whether this is also the case in the cardiac myocyte, neonatal rat cardiac myocytes (NRCM) were infected with a recombinant adenovirus expressing a constitutively active mutant of Akt1 (myrAkt1) and then with or without adenoviruses expressing the active LKB1 complex. Expression of myrAkt1 blunted LKB1-induced phosphorylation of AMPKalpha at Thr(172), which resulted in a dramatic decrease in phosphorylation of AMPK's target, acetyl CoA-carboxylase. This decrease in AMPK activity was associated with prior Akt1-dependent phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491). To investigate whether Akt1 activation was also able to prevent other AMPKKs from phosphorylating AMPKalpha, we subjected NRCM to chemical hypoxia and noted a marked increase in phosphorylation of AMPKalpha at Thr(172), despite no change in LKB1 activity. NRCM expressing myrAkt1 demonstrated increased phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491) and a complete inhibition of chemical hypoxia-induced phosphorylation of AMPKalpha at Thr(172). Taken together, our data show that activation of Akt1 is able to prevent activation of cardiac AMPK by LKB1 and at least one other AMPKK, likely by prior phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491).     

High‐throughput cloning and expression library creation for functional proteomics

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single‐gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high‐throughput proteomics platforms, such as protein microarrays and cell‐based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high‐throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator^TM DNA Cloning System) and compare them side‐by‐side. We also report an example of high‐throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12).     

Molecular assays for detecting Aphanomyces invadans in ulcerative mycotic fish lesions

The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans.     

Recurrent Tissue-Specific mtDNA Mutations Are Common in Humans

Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in two additional individuals. These recurrent mutations all occur within or in very close proximity to sites that regulate mtDNA replication, strongly implying that these variations alter the replication dynamics of the mutated mtDNA genome. These recurrent variants are all independent of each other and do not occur in the mtDNA coding regions. The most parsimonious explanation of the data is that these frequently repeated mutations experience tissue-specific positive selection, probably through replication advantage.     

Evidence for a dinuclear active site in the metallo-beta-lactamase BcII with substoichiometric Co(II). A new model for metal uptake

Metallo-beta-lactamases are zinc-dependent enzymes that constitute one of the main resistance mechanisms to beta-lactam antibiotics. Metallo-beta-lactamases have been characterized both in mono- and dimetallic forms. Despite many studies, the role of each metal binding site in substrate binding and catalysis is still unclear. This is mostly due to the difficulties in assessing the metal content and site occupancy in solution. For this reason, Co(II) has been utilized as a useful probe of the active site structure. We have employed UV-visible, EPR, and NMR spectroscopy to study Co(II) binding to the metallo-beta-lactamase BcII from Bacillus cereus. The spectroscopic features were attributed to the two canonical metal binding sites, the 3H (His(116), His(118), and His(196)) and DCH (Asp(120), Cys(221), and His(263)) sites. These data clearly reveal the coexistence of mononuclear and dinuclear Co(II)-loaded forms at Co(II)/enzyme ratios as low as 0.6. This picture is consistent with the macroscopic dissociation constants here determined from competition binding experiments. A spectral feature previously assigned to the DCH site in the dinuclear species corresponds to a third, weakly bound Co(II) site. The present work emphasizes the importance of using different spectroscopic techniques to follow the metal content and localization during metallo-beta-lactamase turnover.