Genetic analysis of Pycr1 and Pycr2 in mice

The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

Degradation of extracellular matrix by the trophoblastic cells of first-trimester human placentas

First-trimester human placental villi were cultured on 3H-leucine-labeled extracellular matrices isolated from the PF HR9 and PYS-2 cell lines. Both cell lines produced an extracellular matrix that contained basement membrane-specific macromolecules, including type IV collagen, laminin and proteoglycan. Both matrices promoted outgrowth of cells from the villi which, according to morphological criteria, were identified as cytotrophoblastic cells. As the cells migrated from the attachment site, they caused a marked focal dissolution of the matrix which was accompanied by a concomitant release of 3H-labeled material into the media. Approximately half of this material chromatographed near the inclusion volume of Sephadex G-50, indicating that the labeled matrix components had been degraded. This phenomenon was dependent on the age of the placenta. Second-trimester placental villi also adhered to the matrix, but no areas of dissolution were formed and no significant amounts of radioactivity were released into the medium. These results suggest that culture of first-trimester human placental villi on extracellular matrices may be useful for the study of some of the early embryonic events leading to human implantation, during which the trophoblastic cells erode the uterine epithelium.     

Comparison of soluble starch synthases and branching enzymes from developing maize and teosinte seeds

Soluble starch synthases and branching enzymes were purified from developing seeds of the maize inbreds W64A and Ia5125, annual teosintes cv. Galinat's Northern Teosinte and race “Nobogame” and diploid perennial teosinte. Two fractions of starch synthase were obtained by DEAE-cellulose chromatography in all purifications. Starch synthase I fractions had citrate stimulated activity and were most active in primed reactions containing glucogen. Starch synthase II fractions were more active in primed reactions with amylopectine and showed no citrate stimulated activity. Three fractions of branching enzymes were similar kinetically and chromatographically. In addition, antibodies prepared against maize branching enzymes cross reacted with teosinte enzymes. Precipitation lines double diffusion experiments and similar neutralizations of enzymes. precipitation lines of identity in double diffusion experiments and similar neutralization of enzyme activity wiyh increasing levels of antiserum, support the conclusion that maize and teosinte enzymes are highly homologous.     

Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1

Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.     

Evaluating a science-based decision support tool used to prioritize brook charr conservation project proposals in the eastern United States

Declines in brook charr (Salvelinus fontinalis) throughout its historic eastern United States of America (U.S.) range prompted the formation of the Eastern Brook Trout [Charr] Joint Venture (Joint Venture) whose mandate is to restore these charr by working cooperatively at a range-wide, regional-scale. Joint Venture habitat projects in the U.S. are initiated by local community-based organizations, assisted by fisheries management institutions, and funded through the U.S. Fish and Wildlife Service. In order to assist with ranking these community-based proposals for available funding, the Joint Venture designed a quantitative scoring method as a decision support tool that addressed range-wide threats that were documented to limit brook charr production. These threats include habitat modification (e.g., dams, urbanization, and agricultural landuse) and interactions with antagonistic non-native species [i.e., brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss)]. This article evaluated the prioritization method used by comparing the quantitative rankings of the proposals with the final reports for 12 completed projects using paired t tests for unequal sample sizes and variance. Two of the 12 comparisons revealed statistically significant differences (P < 0.05) between project proposals and final reports; namely, the scoring criteria for the proposals did not accurately reflect the outcome of these projects in relation to brook charr management. Large inter-quartile ranges between reviewer scores were common, indicating that the quantitative scoring method includes qualitative elements. In addition to the science-based criteria, subjective value judgments by individual reviewers have factored into the Joint Venture’s decision making process. By evaluating the decision support tool, this analysis aims to assist the Joint Venture in structuring its approach to conservation and rehabilitation of brook charr populations in the eastern U.S. To better improve the Joint Venture’s ability to address brook charr management on a regional level, we recommend that future proposal prioritization scoring criteria provide more explicit guidance for accurate scoring, separate scoring methods for policy and assessment work, and a weighting factor for larger than sub-watershed projects and for scoring criteria for which information is unknown.     

Evolution of isozyme loci and their differential tissue expression

Summary The phylogeny of the creatine kinase (CK, EC 2.7.3.2) isozyme loci and their differential tissue expressions were determined for representatives of 65 families of vertebrates, with emphasis on the fishes. The transition from the single creatine kinase locus, characteristic of certain echinoderms, to the two creatine kinase loci which are orthologous to those present in all vertebrates, occurred early in the chordate line. The majority of pre-teleostean fishes possesses only these two CK loci (A and C). These loci are relatively generalized in their tissue expressions which are variable among species of primitive fishes. The third and fourth creatine kinase loci (B and D) arose separately in the ancestors of the bony fishes and appear to be the result of regional genome duplications. Concomitant with the increase in the number of isozyme loci has been an increase in the specificity of their tissue expression. In the advanced teleost fishes the four CK loci are differentially expressed in a characteristic manner. The A₂ isozyme predominates in skeletal muscle, the B₂ isozyme in eye and brain, the C₂ isozyme in stomach muscle, and the D₂ isozyme is found exclusively in testis. We propose a phylogeny of the creatine kinase genes in the lower chordates based on the time of appearance of new CK loci, the sequence in which the loci achieve a tissue restricted expression, and the immunochemical relatedness of the orthologous and paralogous gene products.     

Larger than Life: Humans' Nonverbal Status Cues Alter Perceived Size

Background

Social dominance and physical size are closely linked. Nonverbal dominance displays in many non-human species are known to increase the displayer''s apparent size. Humans also employ a variety of nonverbal cues that increase apparent status, but it is not yet known whether these cues function via a similar mechanism: by increasing the displayer''s apparent size.

Methodology/Principal Finding

We generated stimuli in which actors displayed high status, neutral, or low status cues that were drawn from the findings of a recent meta-analysis. We then conducted four studies that indicated that nonverbal cues that increase apparent status do so by increasing the perceived size of the displayer. Experiment 1 demonstrated that nonverbal status cues affect perceivers'' judgments of physical size. The results of Experiment 2 showed that altering simple perceptual cues can affect judgments of both size and perceived status. Experiment 3 used objective measurements to demonstrate that status cues change targets'' apparent size in the two-dimensional plane visible to a perceiver, and Experiment 4 showed that changes in perceived size mediate changes in perceived status, and that the cue most associated with this phenomenon is postural openness.

Conclusions/Significance

We conclude that nonverbal cues associated with social dominance also affect the perceived size of the displayer. This suggests that certain nonverbal dominance cues in humans may function as they do in other species: by creating the appearance of changes in physical size.     

Association of Thioautotrophic Bacteria with Deep-Sea Sponges

We investigated microorganisms associated with a deep-sea sponge, Characella sp. (Pachastrellidae) collected at a hydrothermal vent site (686 m depth) in the Sumisu Caldera, Ogasawara Island chain, Japan, and with two sponges, Pachastrella sp. (Pachastrellidae) and an unidentified Poecilosclerida sponge, collected at an oil seep (572 m depth) in the Gulf of Mexico, using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) directed at bacterial 16S rRNA gene sequences. In the PCR-DGGE profiles, we detected a single clearly dominant band in each of the Characella sp. and the unidentified Poecilosclerida sponge. BLAST search of their sequences showed that they were most similar (>99% identity) to those of the gammaproteobacterial thioautotrophic symbionts of deep-sea bivalves from hydrothermal vents, Bathymodiolus spp. Phylogenetic analysis of the near-full length sequences of the 16S rRNA genes cloned from the unidentified Poecilosclerida sponge and Characella sp. confirmed that they were closely related to thioautotrophic symbionts. Although associations between sponges and methanotrophic bacteria have been reported previously, this is the first report of a possible stable association between sponges and thioautotrophic bacteria.