Accumulation of CK-MM is impaired in innervated and contracting cultured muscle fibers of Duchenne muscular dystrophy patients

No specific abnormalities have been reproducibly manifested in aneurally cultured muscle of Duchenne muscular dystrophy (DMD) patients. We now report that the accumulation of the muscle-"specific" isozyme of creatine kinase (CK-MM) was significantly and preferentially impaired in long-term innervated contracting muscle fibers cultured from 4 DMD patients (DMD-InnCMFs) compared to: i) their noninnervated sister-cultured muscle fibers, and ii) innervated contracting control cultured human muscle fibers (Control-InnCHMFs). Accumulation of other muscle-"specific" isozymes (MSIs), viz. glycogen phosphorylase, phosphoglycerate mutase, and lactic dehydrogenase, was not significantly impaired. We have not observed preferentially-impaired CK-MM accumulation in any Control-InnCHMFs from 22 patients (children and adults) with a variety of neuromuscular diseases. There was no apparent difference between DMD-InnCMFs and Control InnCHMFs regarding: acceptance of innervation; neuronally-driven, virtually continuous muscle-fiber contractions; characteristic myofiber organization by phase-contrast microscopy, and increased longevity of the innervated fibers.     

Ectopic activation of Dpp signalling in the male Drosophila germline inhibits germ cell differentiation

The mechanisms that control differentiation of stem cells to specialised cell types probably include factors intrinsic to stem cells as well as extrinsic factors produced by the microenvironment of the stem cell niche. The Drosophila male germline is renewed from a population of stem cells located in the apical tip of the adult testis. The morphological relationship between germline stem cells and their surrounding somatic cells is well understood but the factors that regulate stem cell proliferation and differentiation are still being uncovered. This study examined the effect of stimulating Dpp signalling directly in male germ cells. Ectopic Dpp or Activin signalling resulted in overproliferation of both stem cell-like and spermatogonial-like cells in the apical region of the testis. A third cell population that expressed stem cell markers was seen to proliferate in the distal testis when Dpp signalling was either stimulated or repressed in germline stem cells.     

Conspecific plant–soil feedback scales with population size in Lobelia siphilitica (Lobeliaceae)

Plant–soil interactions directly affect plant success in terms of establishment, survival, growth and reproduction. Negative plant–soil feedback on such traits may therefore reduce the density and abundance of plants of a given species at a given site. Furthermore, if conspecific feedback varies among population sites, it could help explain geographic variation in plant population size. We tested for among-site variation in conspecific plant–soil feedback in a greenhouse experiment using seeds and soils from 8 natural populations of Lobelia siphilitica hosting 30–330 plants. The first cohort of seeds was grown on soil collected from each native site, while the second cohort was grown on the soil conditioned by the first. Our goal was to distinguish site-specific effects mediated by biotic and/or abiotic soil properties from those inherent in seed sources. Cohort 1 plants grown from seeds produced in small populations performed better in terms of germination, growth, and survival compared to plants produced in large populations. Plant performance decreased substantially between cohorts, indicating strong negative feedback. Most importantly, the strength of negative feedback scaled linearly (i.e., was less negative) with increasing size of the native plant population, particularly for germination and survival, and was better explained by soil- rather than seed-source effects. Even with a small number of sites, our results suggest that the potential for negative plant–soil feedback varies among populations of L. siphilitica, and that small populations were more susceptible to negative feedback. Conspecific plant–soil feedback may contribute to plant population size variation within a species’ native range.     

High-level secretion of biologically active aprotinin from the yeastPichia pastoris

Summary A synthetic gene encoding aprotinin (bovine pancreatic trypsin, inhibitor) was fused to theSaccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site.Pichia pastoris strains were developed to'express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion.P. pastoris containing a single copy of the vector secreed approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipoten with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.     

A lateral-displacement flume for fish behavior and stranding studies during simulated pulsed flows

In regulated rivers, fluctuating water depths associated with pulsed discharges may strand small fish in side channels and pools. Quantitative assessments of stranded fish are difficult in field studies (e.g., due to unknown effects of avian and terrestrial vertebrate predators). To assess such lateral displacement and stranding on juvenile stream fishes, we designed, constructed, and tested (with three species) a 2 × 1-m, lateral-displacement flume. The flume featured a main channel that never drained and a raised, wide “floodplain” channel that alternately flooded, with a simulated pulse, and became dewatered. The floodplain contained four pools, with different shapes and draining capacities, in which fish could become stranded as the water level subsided. Fish-stranding rates (8%) in this relatively compact laboratory flume, after exposure to simulated pulsed stream flows, were comparable to those observed in past investigations using larger, artificial streams.     

Manipulation of estrogen synthesis alters MIR202* expression in embryonic chicken gonads

Tissue-specific patterns of microRNA (miRNA) expression contribute to organogenesis during embryonic development. Using the embryonic chicken gonads as a model for vertebrate gonadogenesis, we previously reported that miRNAs are expressed in a sexually dimorphic manner during gonadal sex differentiation. Being male biased, we hypothesised that up-regulation of microRNA 202* (MIR202*) is characteristic of testicular differentiation. To address this hypothesis, we used estrogen modulation to induce gonadal sex reversal in embryonic chicken gonads and analyzed changes in MIR202* expression. In ovo injection of estradiol-17beta at Embryonic Day 4.5 (E4.5) caused feminization of male gonads at E9.5 and reduced MIR202* expression to female levels. Female gonads treated at E3.5 with an aromatase inhibitor, which blocks estrogen synthesis, were masculinized by E9.5, and MIR202* expression was increased. Reduced MIR202* expression correlated with reduced expression of the testis-associated genes DMRT1 and SOX9, and up-regulation of ovary-associated genes FOXL2 and CYP19A1 (aromatase). Increased MIR202* expression correlated with down-regulation of FOXL2 and aromatase and up-regulation of DMRT1 and SOX9. These results confirm that up-regulation of MIR202* coincides with testicular differentiation in embryonic chicken gonads.     

Immune suppression in the tumor microenvironment: a role for dendritic cell-mediated tolerization of T cells

Immune suppression remains a consistent obstacle to successful anti-tumor immune responses. As tumors develop, they create a microenvironment that not only supports tumor growth and metastasis but also reduces potential adaptive immunity to tumor antigens. Among the many components of this tumor microenvironment is a population of dendritic cells which exert profound immune suppressive effects on T cells. In this review, we discuss our recent findings related to these tumor-associated dendritic cells and how targeting them may serve to generate more durable anti-tumor immune responses.     

Kinetic model of the epidermal growth factor (EGF) receptor tyrosine kinase and a possible mechanism of its activation by EGF.

The tyrosine kinase activity of the epidermal growth factor receptor (EGFR-TK) was determined at varying poly-Glu6Ala3Tyr1 (GAT) or [Val5]-angiotensin II (AT) and constant ATP concentrations and vice versa. With GAT as substrate, double reciprocal plots intersected practically on the abscissa following EGFR-TK pre-activation with EGF, but below the abscissa without EGF pre-activation. The EGFR-TK inhibitors App(NH)p (5'-adenylyl-beta, gamma-imidodiphosphate) and ADP were competitive with ATP and noncompetitive with GAT. Four families of 1/v vs. 1/[ATP] plots, constructed at different fixed concentrations of ADP and a different constant concentration of GAT for each family, yielded Slope1/ATP replots which intersected to the left of the ordinate and below the abscissa. GAT and AT, as cosubstrates, were competitive with each other and noncompetitive with ATP; 1/v vs. 1/[GAT] or 1/[AT] plots were hyperbolic and reached horizontal asymptotes when v was expressed as the rate of common product formation. All data were subjected to computer best-fit analysis by a program written especially for this purpose. We conclude that (i) the EGFR-TK reaction follows a Sequential Bi-Bi Rapid Equilibrium Random mechanism, and (ii) EGF induces conformational changes in the EGFR-TK active center which lead to marked decreases in the apparent dissociation constants of both substrates of the kinase reaction and a concomitant increase in initial velocities and Vmax (apparent).     

Organismal differences in post-translational modifications in histones H3 and H4

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.