Molecular cloning and analysis of Staphylococcus aureus chromosomal aminoglycoside resistance genes

Most of the aminoglycoside resistant Staphylococcus aureus strains isolated in France are resistant to all the antibiotics belonging to this family. Two aminoglycoside-modifying enzymes were detected in the wild-type strains studied: an APH3'III and an AAC6'-APH2". These strains also carry two types of streptomycin resistance: high-level resistance due to chromosomal mutation(s) affecting ribosome affinity and low-level resistance, the mechanism of which was not characterized. All the aminoglycoside resistance genes were located on the chromosome. DNA fragments of 1.5 and 1.95 kb carrying the aphA and aacA genes, respectively, were isolated, by cloning, from the cellular DNA of a clinical isolate. When these genes were introduced into Escherichia coli and Bacillus subtilis strains, the enzymes synthesized were indistinguishable from those produced by the S. aureus strains. When the cellular DNAs of wild-type and resistant strains were hybridized with the cloned fragments, sequences homologous to the fragment carrying the aphA gene were found to be located at the same chromosomal site, while those hybridizing with the fragment carrying the aacA gene were at different chromosomal sites.     

The genera in the second catalogue (1833–1836) of?Dejean’s Coleoptera collection

All genus-group names listed in the second edition of the catalogue (1833-1836) of Dejean’s beetle collection are recorded. For each new genus-group name the originally included available species are listed and for generic names with at least one available species, the type species and the current status are given. Names available prior to the publication of Dejean’s second catalogue (1833-1836) are listed in an appendix.The following new synonymies are proposed: Cyclonotum Dejean, 1833 (= Dactylosternum Wollaston, 1854) [Hydrophilidae], Hyporhiza Dejean, 1833 (= Rhinaspis Perty, 1830) [Scarabaeidae], Aethales Dejean, 1834 (= Epitragus Latreille, 1802) [Tenebrionidae], Arctylus Dejean, 1834 (= Praocis Eschscholtz, 1829) [Tenebrionidae], Euphron Dejean, 1834 (= Derosphaerus Thomson, 1858) [Tenebrionidae], Hipomelus Dejean, 1834 (= Trachynotus Latreille, 1828) [Tenebrionidae], Pezodontus Dejean, 1834 (= Odontopezus Alluaud, 1889) [Tenebrionidae], Zygocera Dejean, 1835 (= Disternopsis Breuning, 1939) [Cerambycidae], and Physonota Chevrolat, 1836 (= Anacassis Spaeth, 1913) [Chrysomelidae]. Heterogaster pilicornis Dejean, 1835 [Cerambycidae] and Labidomera trimaculata Chevrolat, 1836 [Chrysomelidae] are placed for the first time in synonymy with Anisogaster flavicans Deyrolle, 1862 and Chrysomela clivicollis Kirby, 1837 respectively. Type species of the following genus-group taxa are proposed: Sphaeromorphus Dejean, 1833 (Sphaeromorphus humeralis Erichson, 1843) [Scarabaeidae], Adelphus Dejean, 1834 (Helops marginatus Fabricius, 1792) [Tenebrionidae], Cyrtoderes Dejean, 1834 (Tenebrio cristatus DeGeer, 1778) [Tenebrionidae], Selenepistoma Dejean, 1834 (Opatrum acutum Wiedemann, 1823) [Tenebrionidae], Charactus Dejean, 1833 (Lycus limbatus Fabricius, 1801) [Lycidae], Corynomalus Chevrolat, 1836 (Eumorphus limbatus Olivier, 1808) [Endomychidae], Hebecerus Dejean, 1835 (Acanthocinus marginicollis Boisduval, 1835) [Cerambycidae], Pterostenus Dejean, 1835 (Cerambyx abbreviatus Fabricius, 1801) [Cerambycidae], Psalicerus Dejean, 1833 (Lucanus femoratus Fabricius, 1775) [Lucanidae], and Pygolampis Dejean, 1833 (Lampyris glauca Olivier, 1790) [Lampyridae]. A new name, Neoeutrapela Bousquet and Bouchard [Tenebrionidae], is proposed for Eutrapela Dejean, 1834 (junior homonym of Eutrapela Hübner, 1809).The following generic names, made available in Dejean’s catalogue, were found to be older than currently accepted valid names: Catoxantha Dejean, 1833 over Catoxantha Solier, 1833 [Buprestidae], Pristiptera Dejean, 1833 over Pelecopselaphus Solier, 1833 [Buprestidae], Charactus Dejean, 1833 over Calopteron Laporte, 1836 [Lycidae], Cyclonotum Dejean, 1833 over Dactylosternum Wollaston, 1854 [Hydrophilidae], Ancylonycha Dejean, 1833 over Holotrichia Hope, 1837 [Scarabaeidae], Aulacium Dejean, 1833 over Mentophilus Laporte, 1840 [Scarabaeidae], Sciuropus Dejean, 1833 over Ancistrosoma Curtis, 1835 [Scarabaeidae], Sphaeromorphus Dejean, 1833 over Ceratocanthus White, 1842 [Scarabaeidae], Psalicerus Dejean, 1833 over Leptinopterus Hope, 1838 [Lucanidae], Adelphus Dejean, 1834 over Praeugena Laporte, 1840 [Tenebrionidae], Amatodes Dejean, 1834 over Oncosoma Westwood, 1843 [Tenebrionidae], Cyrtoderes Dejean, 1834 over Phligra Laporte, 1840 [Tenebrionidae], Euphron Dejean, 1834 over Derosphaerus Thomson, 1858 [Tenebrionidae], Pezodontus Dejean, 1834 over Odontopezus Alluaud, 1889 [Tenebrionidae], Anoplosthaeta Dejean, 1835 over Prosopocera Blanchard, 1845 [Cerambycidae], Closteromerus Dejean, 1835 over Hylomela Gahan, 1904 [Cerambycidae], Hebecerus Dejean, 1835 over Ancita Thomson, 1864 [Cerambycidae], Mastigocera Dejean, 1835over Mallonia Thomson, 1857 [Cerambycidae], Zygocera Dejean, 1835 over Disternopsis Breuning, 1939 [Cerambycidae], Australica Chevrolat, 1836 over Calomela Hope, 1840 [Chrysomelidae], Edusa Chevrolat, 1836 over Edusella Chapuis, 1874 [Chrysomelidae], Litosonycha Chevrolat, 1836 over Asphaera Duponchel and Chevrolat, 1842 [Chrysomelidae], and Pleuraulaca Chevrolat, 1836 over Iphimeis Baly, 1864 [Chrysomelidae]. In each of these cases, Reversal of Precedence (ICZN 1999: 23.9) or an applicationto the International Commission on Zoological Nomenclature will be necessary to retain usage of the younger synonyms.     

Rapid internalization and recycling of the human neuropeptide Y Y(1) receptor.

Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y(1) receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y(1) receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y(1) receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y(1) receptors. Upon agonist challenge, the EGFP signal decreased rapidly (t(1/2) = 107 +/- 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y(1) receptor antagonist, or by pertussis toxin, in agreement with Y(1) receptor activation. Internalization of EGFP-hNPY Y(1) receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y(1) receptors.     

Cysteine-proteinase-inhibiting function of T kininogen and of its proteolytic fragments

Previous attempts to liberate T kinin from T kininogen [Moreau et al. (1986) Eur. J. Biochem. 159, 341-346; Gutman et al. (1988) Eur. J. Biochem. 171, 577-582] have shown that complete fragmentation of the precursor molecule into inhibitory peptides was achieved before any vasoactive peptide was released, suggesting a possible physiological significance for this phenomenon. In this study, cysteine-proteinase-inhibiting properties of rat T kininogen and of its proteolytic fragments issuing from trypsin and submaxillary gland endopeptidase k hydrolysis, have been investigated using rat lysosomal cathepsins B, H and L, papain and bovine calpains I and II. All three lysosomal cathepsins were inhibited by T kininogen but tighter interactions were observed with cathepsin L and papain. Though higher Ki values were obtained for cathepsins B and H, rate constants for association were found to have high and almost similar values (in the 10(6) M-1 s-1 range) whatever the enzyme used. Proteolytic fragments also inhibited cathepsin L and papain very strongly and even better than the entire molecule for some of them, but no significant inhibition of cathepsins B and H was observed. Bovine calpains were not inhibited by T kininogen nor by its proteolytic fragments. From the results of this kinetic analysis, which indicates that both the association and the dissociation of lysosomal cysteine proteinases with T kininogen may occur rapidly, an hypothesis has been put forward on the possible in vivo functioning of T kininogen as a proteinase inhibitor.     

Defensive traits exhibit an evolutionary trade‐off and drive diversification in ants

Evolutionary biologists have long predicted that evolutionary trade‐offs among traits should constrain morphological divergence and species diversification. However, this prediction has yet to be tested in a broad evolutionary context in many diverse clades, including ants. Here, we reconstruct an expanded ant phylogeny representing 82% of ant genera, compile a new family‐wide trait database, and conduct various trait‐based analyses to show that defensive traits in ants do exhibit an evolutionary trade‐off. In particular, the use of a functional sting negatively correlates with a suite of other defensive traits including spines, large eye size, and large colony size. Furthermore, we find that several of the defensive traits that trade off with a sting are also positively correlated with each other and drive increased diversification, further suggesting that these traits form a defensive suite. Our results support the hypothesis that trade‐offs in defensive traits significantly constrain trait evolution and influence species diversification in ants.     

Naf1p,an essential nucleoplasmic factor specifically required for accumulation of box H/ACA small nucleolar RNPs

Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. The ways in which these particles are assembled and correctly localized in the dense fibrillar component of the nucleolus remain largely unknown. Recently, the essential Saccharomyces cerevisiae Naf1p protein (encoded by the YNL124W open reading frame) was found to interact in a two-hybrid assay with two core protein components of mature H/ACA snoRNPs, Cbf5p and Nhp2p (T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, Proc. Natl. Acad. Sci. USA 98:4569-4574, 2001). Here we show that several H/ACA snoRNP components are weakly but specifically immunoprecipitated with epitope-tagged Naf1p, suggesting that the latter protein is involved in H/ACA snoRNP biogenesis, trafficking, and/or function. Consistent with this, we find that depletion of Naf1p leads to a defect in 18S rRNA accumulation. Naf1p is unlikely to directly assist H/ACA snoRNPs during pre-rRNA processing in the dense fibrillar component of the nucleolus for two reasons. Firstly, Naf1p accumulates predominantly in the nucleoplasm. Secondly, Naf1p sediments in a sucrose gradient chiefly as a free protein or associated in a complex of the size of free snoRNPs, whereas extremely little Naf1p is found in fractions containing preribosomes. These results are more consistent with a role for Naf1p in H/ACA snoRNP biogenesis and/or intranuclear trafficking. Indeed, depletion of Naf1p leads to a specific and dramatic decrease in the steady-state accumulation of all box H/ACA snoRNAs tested and of Cbf5p, Gar1p, and Nop10p. Naf1p is unlikely to be directly required for the synthesis of H/ACA snoRNP components. Naf1p could participate in H/ACA snoRNP assembly and/or transport.     

Spring phenology at different altitudes is becoming more uniform under global warming in Europe

Under current global warming, high‐elevation regions are expected to experience faster warming than low‐elevation regions. However, due to the lack of studies based on long‐term large‐scale data, the relationship between tree spring phenology and the elevation‐dependent warming is unclear. Using 652k records of leaf unfolding of five temperate tree species monitored during 1951–2013 in situ in Europe, we discovered a nonlinear trend in the altitudinal sensitivity (S_A, shifted days per 100 m in altitude) in spring phenology. A delayed leaf unfolding (2.7 ± 0.6 days per decade) was observed at high elevations possibly due to decreased spring forcing between 1951 and 1980. The delayed leaf unfolding at high‐elevation regions was companied by a simultaneous advancing of leaf unfolding at low elevations. These divergent trends contributed to a significant increase in the S_A (0.36 ± 0.07 days 100/m per decade) during 1951–1980. Since 1980, the S_A started to decline with a rate of ?0.32 ± 0.07 days 100/m per decade, possibly due to reduced chilling at low elevations and improved efficiency of spring forcing in advancing the leaf unfolding at high elevations, the latter being caused by increased chilling. Our results suggest that due to both different temperature changes at the different altitudes, and the different tree responses to these changes, the tree phenology has shifted at different rates leading to a more uniform phenology at different altitudes during recent decades.     

Progress in outgrowth culture from rabbit tracheal explants: Balance between proliferation and maintenance of differentiated state in epithelial cells

Summary Primary cultures of rabbit tracheal cells were obtained as outgrowths from explants of tracheal mucosa. A 30% collagen substratum containing serum and minimal essential medium was required for obtaining an outgrowth of epithelial cells keeping their differentiated characteristics. The tracheal epithelial cells obtained near the explant in the first days of culture presented morphologic similarities with normal tracheal epithelium. Cultures contained basal cells and epithelial polarized cells that exhibited apical tight junctions and desmosomes. Ciliated cells stayed functional during all time culture. Their number slightly increased at the beginning of the culture and then stayed constant when the total number of cells increased. Development of the outgrowth was rapid and significant inasmuch as the outgrowth surface reached 30 times that of the explant after less than 8 days. This was linked to cellular proliferation, as demonstrated by the incorporation of bromodeoxyuridine (BrdU) in phase-S nuclei and the revelation of BrdU using an immunofluorescence technique. The epithelial nature of the outgrowth cells and the absence of contamination with fibroblasts were established by positive staining with anti-keratin antibody and by negative staining with anti-vimentin antibody, respectively. This work was supported by DRET and by CIFRE grant awarded to S. R.