Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.     

Immunocytochemical identification of proctolinlike immunoreactivity in the terminal ganglion and hindgut of the cockroach Periplaneta americana (L.)

Summary In the American cockroach, the distribution and connections of neuronal elements of the terminal ganglion-proctodeal nerve-hindgut system were investigated by means of immunohistochemical methods and axonal CoCl₂ iontophoresis. Proctolinlike immunoreactivity was localized within neurons of the terminal ganglion projecting into the proctodeal nerve on the one hand, and in nerve cells without a direct connection to this system on the other. Immunohistochemically, in whole mount preparations fibres of the proctodeal nerve and terminal structures in the hindgut musculature exhibit strong proctolinlike immunoreactivity. At the light- and electron-microscopic levels the pathways of about 30 somata of the proctodeal neural system were characterized by cobalt chloride iontophoresis. The relationships of cobalt filled and immunoreactive neuronal structures are discussed.For the preparation of tritiated proctolin we thank Dr. S. Reißmann, WB Biochemie, Sektion Biologie, FSU JenaThe authors wish to thank G. Schörlitz, Film- und Bildstelle, FSU Jena, for photographs of whole mount preparations and Ms. A. Zinßer and Mrs. B. Cosack for excellent technical assistance.     

Plasmodium cynomolgi: the hsp 70 gene.

The hsp 70 gene of Plasmodium cynomolgi was isolated and characterized. As expected the gene is highly similar to that of the hsp 70 gene of Plasmodium falciparum (98% at the protein level, 82% at the nucleotide level). Surprisingly, the hsp 70 gene appears to be present in a single copy in all the P. cynomolgi strains tested, a finding that has implications for the parasite's ability to undergo a heat shock response.     

Phosphorylation modulates keratin structure.

Bovine hoof keratin was shown to be a substrate for cAMP-dependent protein kinase using [gamma-32P]ATP. Natural-abundance cross-polarization (CP) MAS 13C NMR was used to examine the effect of phosphorylation on keratin structure. When short contact times were used, phosphorylation was shown to increase the number of residues in the motionally restricted portions of the protein; i.e., a portion(s) of the protein became more rigid upon phosphorylation. Circular dichroism (CD) spectra showed a spectral shape characteristic of alpha helix for this keratin. Phosphorylation of the keratin by cAMP-dependent protein kinase resulted in a CD spectrum with the same shape but of greater apparent intensity. This may have been the result of an increase in the alpha-helical content of the protein. These data showed that the structure of keratin changed significantly upon phosphorylation by cAMP-dependent protein kinase. The region of the keratin molecule most likely to be altering its structure was the end of the molecule, which was involved in the formation of, and intracellular attachment of, intermediate filaments. Therefore, these data suggested that cAMP-dependent phosphorylation may produce significant changes in the intracellular organization of intermediate filaments. When the keratin was phosphorylated using cold ATP, magic-angle spinning (MAS) 31P nuclear magnetic resonance (NMR) revealed two resonances arising from the phosphorylation sites on the keratin. The more shielded resonance was shown to arise from cAMP-dependent protein kinase phosphorylation. Static 31P NMR measurements suggested that at least two classes of cAMP-dependent sites existed with the same isotropic 31P chemical shift; one was considerably motionally restricted with respect to the other.     

Assembly of keratin onto PtK1 cytoskeletons: evidence for an intermediate filament organizing center

Purified keratin, solubilized in 8 M of urea, was added to Triton X-100-extracted PtK1 cells in 5 mM PIPES buffer. The buffer conditions induced assembly of keratin filaments which appear to associate with nuclei of extracted cells. These keratin fibers extend beyond the original margin of the cells and frequently form bridges between adjacent cells. Electron microscopy shows that keratin filaments associate closely with the surface of the nucleus. We suggest that the site of association between keratin and the nucleus may represent an intermediate filament organizing center.     

Electron microscopical-immunocytochemical evidence of ecdysteroids in the prothoracic gland of Galleria mellonella

Summary Fixation of prothoracic glands of Galleria mellonella with a solution containing saponin permits immunocytochemical staining of the entire gland. By this means ecdysteroids were demonstrated electron microscopically to be present in the hyaloplasm and microtubules.Supported by Sächsische Akademie der Wissenschaften zu Leipzig     

Genotyping‐by‐sequencing and ecological niche modeling illuminate phylogeography,admixture, and Pleistocene range dynamics in quaking aspen (Populus tremuloides)

Populus tremuloides is the widest‐ranging tree species in North America and an ecologically important component of mesic forest ecosystems displaced by the Pleistocene glaciations. Using phylogeographic analyses of genome‐wide SNPs (34,796 SNPs, 183 individuals) and ecological niche modeling, we inferred population structure, ploidy levels, admixture, and Pleistocene range dynamics of P. tremuloides, and tested several historical biogeographical hypotheses. We found three genetic lineages located mainly in coastal–Cascades (cluster 1), east‐slope Cascades–Sierra Nevadas–Northern Rockies (cluster 2), and U.S. Rocky Mountains through southern Canadian (cluster 3) regions of the P. tremuloides range, with tree graph relationships of the form ((cluster 1, cluster 2), cluster 3). Populations consisted mainly of diploids (86%) but also small numbers of triploids (12%) and tetraploids (1%), and ploidy did not adversely affect our genetic inferences. The main vector of admixture was from cluster 3 into cluster 2, with the admixture zone trending northwest through the Rocky Mountains along a recognized phenotypic cline (Utah to Idaho). Clusters 1 and 2 provided strong support for the “stable‐edge hypothesis” that unglaciated southwestern populations persisted in situ since the last glaciation. By contrast, despite a lack of clinal genetic variation, cluster 3 exhibited “trailing‐edge” dynamics from niche suitability predictions signifying complete northward postglacial expansion. Results were also consistent with the “inland dispersal hypothesis” predicting postglacial assembly of Pacific Northwestern forest ecosystems, but rejected the hypothesis that Pacific‐coastal populations were colonized during outburst flooding from glacial Lake Missoula. Overall, congruent patterns between our phylogeographic and ecological niche modeling results and fossil pollen data demonstrate complex mixtures of stable‐edge, refugial locations, and postglacial expansion within P. tremuloides. These findings confirm and refine previous genetic studies, while strongly supporting a distinct Pacific‐coastal genetic lineage of quaking aspen.     

Growth factor transgenes interactively regulate articular chondrocytes

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin‐like growth factor I (IGF‐I), fibroblast growth factor‐2 (FGF‐2), transforming growth factor beta1 (TGF‐β1), bone morphogenetic protein‐2 (BMP‐2), and bone morphogenetic protien‐7 (BMP‐7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF‐I and FGF‐2 maximized cell proliferation. The three‐transgene group encoding IGF‐I, BMP‐2, and BMP‐7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell‐based articular cartilage repair. J. Cell. Biochem. 114: 908–919, 2013. © 2012 Wiley Periodicals, Inc.