Cell Wall Amidase AmiC1 Is Required for Cellular Communication and Heterocyst Development in the Cyanobacterium Anabaena PCC 7120 but Not for Filament Integrity

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the Gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.     

How to monitor elusive lizards: comparison of capture–recapture methods on giant day geckos (Gekkonidae, Phelsuma madagascariensis grandis) in the Masoala rainforest exhibit, Zurich Zoo

Rapid and reliable estimation of population size is needed for the efficient monitoring of animal populations of conservation concern. Unfortunately, technical advances in this area have not been paralleled in uptake in conservation, which may be due to difficulties in implementation or the lack of general guidelines for application. Here we tested five different methods used to estimate population size [capture–mark–recapture (CMR), finite-mixture models, model averaging of finite-mixture models, accumulation curve methods (ACM), and the line transect method (LT)] using extensive capture–recapture data of the giant day gecko (Gekkonidae, Phelsuma madagascariensis grandis, Gray 1870) at the Masoala rainforest exhibit, Zurich Zoo. When the complete data were analyzed [30 sessions (and 27 sessions for the LT)], all methods except the LT produced similar estimates of population size. The simple ACM gave a small coefficient of variation (CV), but did not cover the most likely value of population size at moderate sampling effort. Nevertheless, the ACM was the only method that showed a reasonable convergence when subsets of data were used. CMR and Pledger models included the reference value in their confidence intervals (CI) after 25 and 30 sessions, respectively. Although model averaging did slightly improve the estimate, the CV was still high for the full dataset. Our method of using subsets of data to test the robustness of estimates is simple to apply and could be adopted more widely in such analyzes to evaluate sensitivity to method of evaluation. In conclusion, simple accumulation methods showed similar efficiency to more complex statistical models, and are likely to be sufficiently precise for most conservation monitoring purposes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distribution and characterization of the opioid octapeptide met5-enkephalin-arg6-gly7-leu8 in the gastrointestinal tract of the rat

Summary The distribution and characterization of the opioid octapeptide met⁵-enkephalin-arg⁶-gly⁷-leu⁸ (met⁵-enk-arg⁶-gly⁷-leu⁸) within the gastrointestinal tract of the rat has been determined by immunohistochemistry and radioimmunoassay by use of a newly developed antibody to met⁵-enk-arg⁶-gly⁷-leu⁸. With both techniques, met⁵-enk-arg⁶-gly⁷-leu⁸-immunoreactivity (met⁵-enk-arg⁶-gly⁷-leu⁸IR) was detected in all regions of the gastrointestinal (GI) tract except the esophagus. The highest concentration of immunoreactive met⁵-enk-arg⁶-gly⁷-leu⁸ was observed in the colon, while intermediate concentrations were found in the stomach, duodenum, jejunum, and ileum. Immunostained somata were observed chiefly in the myenteric plexus; immunostained processes were present primarily in the myenteric plexus and the circular muscle layer. This distribution pattern is similar to that previously observed with antiserum to met⁵-enkephalin-arg⁶-phe⁷ (met⁵-enk-arg⁶phe⁷). Chromatographic analysis of met⁵-enk-arg⁶-gly⁷leu⁸-immunoreactive peptides extracted from the GI tract revealed the presence of an immunoreactive peptide of high molecular weight which accounted for approximately three-quarters of met⁵-enk-arg⁶-gly⁷-leu⁸-IR in both stomach and colon. These findings suggest a role for peptides related to the octapeptide met⁵-enk-arg⁶-gly⁷-leu⁸ in the regulation of GI function.     

Structural insights into the interaction between prion protein and nucleic acid

The infectious agent of transmissible spongiform encephalopathies (TSE) is believed to comprise, at least in part, the prion protein (PrP). Other molecules can modulate the conversion of the normal PrP(C) into the pathological conformer (PrP(Sc)), but the identity and mechanisms of action of the key physiological factors remain unclear. PrP can bind to nucleic acids with relatively high affinity. Here, we report small-angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy measurements of the tight complex of PrP with an 18 bp DNA sequence. This double-stranded DNA sequence (E2DBS) binds with nanomolar affinity to the full-length recombinant mouse PrP. The SAXS data show that formation of the rPrP-DNA complex leads to larger values of the maximum dimension and radius of gyration. In addition, the SAXS studies reveal that the globular domain of PrP participates importantly in the formation of the complex. The changes in NMR HSQC spectra were clustered in two major regions: one in the disordered portion of the PrP and the other in the globular domain. Although interaction is mediated mainly through the PrP globular domain, the unstructured region is also recruited to the complex. This visualization of the complex provides insight into how oligonucleotides bind to PrP and opens new avenues to the design of compounds against prion diseases.     

Ecotoxicological Evaluation of a Bench-Scale Bioslurry Treating Explosives-Spiked Soil

The ecotoxicological effects of four bioslurry reactors treating 2,4,6-trinitotoluene (TNT)- and 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX)-spiked soil were evaluated. A control bioslurry reactor was used to assess the endogenous toxicity of the bioslurry operation conditions. A battery of ecotoxicity tests was used: Microtox, green algae growth inhibition, bacterial genotoxicity and mutagenicity, and earthworm mortality and growth inhibition. Bioslurry soluble and solid phases were separated by centrifugation in order to identify toxicity and possible toxicants associated with each phase. Microtox toxicity values were initially very high in both bioslurry reactors spiked with TNT, in relation with TNT concentration. Initial toxicity was also detected by algal growth inhibition, earthworm lethality, genotoxicity and mutagenicity tests. An endogenous toxicity was detected in the control bioreactor using the Microtox and the SOS Chromotest. The soluble phase of the control bioslurry was genotoxic, suggesting that some potentially genotoxic agents were induced in the bioslurry samples. At the end of the bioremediation treatment, data showed that toxicity was reduced using all of the bioassays, except for earthworm lethality and growth inhibition tests in both RDX-spiked bioslurries. This study demonstrates the usefulness of a battery of toxicity tests to monitor bioremediation processes.     

Characterization of Enkephalin Release from Rat Striatum

Abstract: Using antisera specific for methionine- and leucine-enkephalin, we studied the characteristics of the release of these peptides from rat striatal slices. Only 2–3% of the total tissue stores of enkephalin could be released by potassium depolarization; similar percentages were released from globus pallidus, thalamus, and nucleus accumbens. Enkephalin release from hippocampus could not be detected. The striatal release of both enkephalins was affected similarly by changes in potassium and calcium levels in the superfusion medium. Lithium has no effect on either basal or potassium-stimulated release; tyr-arg did not affect basal release of either peptide. Striatal enkephalin levels were stable during the short-term incubation periods used in these experiments.     

Dual location of MAR-binding, filament-like protein 1 in Arabidopsis, tobacco, and tomato

Matrix attachment region-binding filament-like protein 1 (MFP1) is a plant-specific long coiled-coil protein that binds double-stranded DNA. While originally identified as a component of the tobacco nuclear matrix, it was subsequently shown that the majority of MFP1 resides in mature chloroplast where it is located at the stroma side of the thylakoids and is able to bind to nucleoids. On the other hand, a 90 kDa MFP1-like protein from onion has been convincingly shown to be an intrinsic component of the onion meristematic nuclear matrix. Here, we have expanded the analysis of the subcellular location of MFP1 by using high-resolution confocal immunofluorescence microscopy and immunogold electron microscopy. Two different antisera raised against MFP1 from two species were used on isolated nuclei and chloroplasts from tomato, tobacco, and Arabidopsis. Our data show that both antibodies detect a signal in both compartments in all three species. An Arabidopsis MFP1 T-DNA insertional mutation abolishes both nuclear and chloroplast signals, indicating that the nuclear and plastidic antigens are derived from the same gene. We therefore suggest that MFP1 is a protein with a dual location, in both nuclei and chloroplasts, consistent with prior findings in onion and the dicot species investigated here.