Characterization of two beta-1,3-glucosyltransferases from Escherichia coli serotypes O56 and O152

The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-beta1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-alpha-PO3-PO3-(CH2)11-O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-beta1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid beta-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn2+ or Mg2+) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.     

UDP-Gal: GlcNAc-R β1,4-galactosyltransferase—a target enzyme for drug design. Acceptor specificity and inhibition of the enzyme

Galactosyltransferases are important enzymes for the extension of the glycan chains of glycoproteins and glycolipids, and play critical roles in cell surface functions and in the immune system. In this work, the acceptor specificity and several inhibitors of bovine β1,4-Gal-transferase T1 (β4GalT, EC 2.4.1.90) were studied. Series of analogs of N-acetylglucosamine (GlcNAc) and GlcNAc-carrying glycopeptides were synthesized as acceptor substrates. Modifications were made at the 3-, 4- and 6-positions of the sugar ring of the acceptor, in the nature of the glycosidic linkage, in the aglycone moiety and in the 2-acetamido group. The acceptor specificity studies showed that the 4-hydroxyl group of the sugar ring was essential for β4GalT activity, but that the 3-hydroxyl could be replaced by an electronegative group. Compounds having the anomeric β-configuration were more active than those having the α-configuration, and O-, S- and C-glycosyl compounds were all active as substrates. The aglycone was a major determinant for the rate of Gal-transfer. Derivatives containing a 2-naphthyl aglycone were inactive as substrates although quinolinyl groups supported activity. Several compounds having a bicyclic structure as the aglycone were found to bind to the enzyme and inhibited the transfer of Gal to control substrates. The best small hydrophobic GlcNAc-analog inhibitor was found to be 1-thio-N-butyrylGlcNβ-(2-naphthyl) with a K_i of 0.01 mM. These studies help to delineate β4GalT–substrate interactions and will aid in the development of biologically applicable inhibitors of the enzyme.     

Temperature responses of macroalgae from the tropical island Hainan (P.R. China)

The temperature tolerances of 24 tropical macroalgae collected on Hainan Island (P.R. China) were investigated. For some isolates, growth response curves were also determined. The upper survival temperatures (USTs, 32–37°C) of these tropical west Pacific strains are similiar to those of tropical Atlantic species. With regard to their lower survival temperatures (LSTs) the species investigated show high variations: 12 species have LSTs between 16 and 7°C (Hypnea musciformis (Wulfen) Lamx. var esperi J, Ag., Centroceras clavulatum (C. Ag) Mont., Falkenbergia hillebrandii (Bornet) Falkenberg, Gelidiopsis intricata (Ag.) Vickers, Halymenia maculata J. Ag., Hypnea cenomyce J. Ag., Hypnea spinella (C. Ag.) Kütz., Gracilaria changii (Xia et Abott) Abott, Chang et Xia, Dictyopteris repens (Okam.) Boerg., Laurencia cartilaginea Yamada, Gelidium pusillum (Stackh.) Le Jol., Laurencia sp.). Their LSTs and temperature requirements for growth (range: 15–30 °C, optimum: 25–30 °C) are mostly similar to those of tropical west Atlantic and amphi-Atlantic (sub)tropical macroalgae as well as to tropical isolates of species with an Atlantic tropical to warm-temperate distribution. The remaining 12 species have LSTs between 6 and 1 °C (Ulva conglobata Kjellm., Ulva fasciata Delile, Padina boryana Thivy, Dictyosphaeria cavernosa (Forssk.) Boerg., Boodlea composita (Harv.) Brand, Boergesenia forbesii (Harv.) Feldm., Cladophora vagabunda (L.) van den Hoek, Enteromorpha compressa (L,) Grev., Enteromorpha intestinalis (L.) Link, Gracilaria tenuistipitata Chang et Xia, var liui Chang et Xia, Monostroma nitidum Wittr. and Valonia aegagropila C. Ag.). Their LSTs are mostly similar to those of Atlantic macroalgae with a tropical to (warm-) temperate distribution. The results are discussed with respect to the factors which may have triggered the development of the temperature requirements of the various species.     

The phosphomimetic mutation of an evolutionarily conserved serine residue affects the signaling properties of Rho of plants (ROPs)

Plant ROP (Rho of plants) proteins form a unique subgroup within the family of Rho-type small G-proteins of eukaryotes. In this paper we demonstrate that the phosphomimetic mutation of a serine residue conserved in all Rho proteins affects the signaling properties of plant ROPs. We found that the S74E mutation in Medicago ROP6 and Arabidopsis ROP4 prevented the binding of these proteins to their plant-specific upstream activator the plant-specific ROP nucleotide exchanger (PRONE)-domain-containing RopGEF (guanine nucleotide exchange factor) protein and abolished the PRONE-mediated nucleotide exchange reaction in vitro. Structural modeling supported the hypothesis that potential phosphorylation of the S74 residue interferes with the binding of the PRONE-domain to the adjacent plant-specific R76 residue which plays an important role in functional ROP-PRONE interaction. Moreover, we show that while the binding of constitutively active MsROP6 to the effector protein RIC (ROP-interactive CRIB-motif-containing protein) was not affected by the S74E mutation, the capability of this mutated protein to bind and activate the RRK1 kinase in vitro was reduced. These observations are in agreement with the morphology of tobacco pollen tubes expressing mutant forms of yellow fluorescent protein (YFP):MsROP6. The S74E mutation in MsROP6 had no influence on pollen tube morphology and attenuated the phenotype of a constitutively active form of MsROP6. The presented Medicago and Arabidopsis data support the notion that the phosphorylation of the serine residue in ROPs corresponding to S74 in Medicago ROP6 could be a general principle for regulating ROP activation and signaling in plants.     

Diagnosis and classification of disorders specifically associated with stress: proposals for ICD‐11

The diagnostic concepts of post‐traumatic stress disorder (PTSD) and other disorders specifically associated with stress have been intensively discussed among neuro‐ and social scientists, clinicians, epidemiologists, public health planners and humanitarian aid workers around the world. PTSD and adjustment disorder are among the most widely used diagnoses in mental health care worldwide. This paper describes proposals that aim to maximize clinical utility for the classification and grouping of disorders specifically associated with stress in the forthcoming 11th revision of the International Classification of Diseases (ICD‐11). Proposals include a narrower concept for PTSD that does not allow the diagnosis to be made based entirely on non‐specific symptoms; a new complex PTSD category that comprises three clusters of intra‐ and interpersonal symptoms in addition to core PTSD symptoms; a new diagnosis of prolonged grief disorder, used to describe patients that undergo an intensely painful, disabling, and abnormally persistent response to bereavement; a major revision of “adjustment disorder” involving increased specification of symptoms; and a conceptualization of “acute stress reaction” as a normal phenomenon that still may require clinical intervention. These proposals were developed with specific considerations given to clinical utility and global applicability in both low‐ and high‐income countries.     

The marine macroalgae of Helgoland (North Sea): an annotated list of records between 1845 and 1999

The earliest known records of marine macroalgae from Helgoland (German Bight, North Sea) date from the mid-19th century. Since then, 274 marine macroalgal species have been reported: 77 species of Chlorophycota, 100 species of Phaeophycota and 97 species of Rhodophycota. Additionally 11 species were only recorded as drift and 51 species as doubtful for Helgoland. The remains of the herbarium of Paul Kuckuck, the first curator for botany at the Helgoland Biological Station between 1892 and 1914, are still located there and consist of 173 macroalgal species from Helgoland. On comparing this 100-year-old herbarium and other old sources with recent macroalgal records, it became clear that changes in species composition have occurred. After World War II, several species such as Arthrocladia villosa, Corynophlaea crispa, Cutleria multifida, Eudesme virescens, Mesogloia vermiculata, Sporochnus pedunculatus, Antithamnion cruciatum, Apoglossum ruscifolium, Chondria dasyphylla, Helminthora divaricata, Jania rubens and Osmundea ramosissima were not found again. Other species such as Dictyota dichotoma, Leathesia difformis, Stictyosiphon soriferus, Helminthocladia calvadosii and Scinaia furcellata became very rare. Significantly, perhaps, most of these species have a heteromorphic life history with the appearance of the macroscopic phase restricted to (spring and) summer. Many new species of green algae were recorded for Helgoland after 1959, due to new substrata and the research activities of Peter Kornmann, curator for botany after 1959, and Paul-Heinz Sahling his technical assistant. Introductions of species during the considered time period were: Bonnemaisonia hamifera, Codium fragile, Mastocarpus stellatus and Sargassum muticum. Type material of the following species is located at the Marine Biological Station at Helgoland: Mikrosyphar porphyrae, Porphyra insolita and Ulva tenera. Received in revised form: 22 May 2000 Electronic Publication     

Interspecific interactions influence contrasting spatial genetic structures in two closely related damselfly species

Spatial genetic structure (SGS) is largely determined by colonization history, landscape and ecological characteristics of the species. Therefore, sympatric and ecologically similar species are expected to exhibit similar SGSs, potentially enabling prediction of the SGS of one species from that of another. On the other hand, due to interspecific interactions, ecologically similar species could have different SGSs. We explored the SGSs of the closely related Calopteryx splendens and Calopteryx virgo within Finland and related the genetic patterns to characteristics of the sampling localities. We observed different SGSs for the two species. Genetic differentiation even within short distances in C. splendens suggests genetic drift as an important driver. However, we also observed indication of previous gene flow (revealed by a negative relationship between genetic differentiation and increasing potential connectivity of the landscape). Interestingly, genetic diversity of C. splendens was negatively related to density of C. virgo, suggesting that interspecific interactions influence the SGS of C. splendens. In contrast, genetic differentiation between C. virgo subpopulations was low and only exhibited relationships with latitude, pointing to high gene flow, colonization history and range margin effects as the drivers of SGS. The different SGSs of the two ecologically similar species caution indirect inferences of SGS based on ecologically similar surrogate species.     

Induction of male sex behavior in pony mares with testosterone propionate

Two pony mares were administered 150 mg of testosterone propionate every other day for 20 days (ten injections) and every ten days there-after. An additional two mares and one stallion were not treated and served as controls. Testosterone propionate was dissolved in absolute ethanol and administered subcutaneously. Sex behavior tests were conducted 26 and 40 days after the first injection. Control mares exhibited very little male sex behavior. Both testosterone propionatetreated mares, however, exhibited mounting, sniffing, flehmen, biting and vocalization behavior in the presence of an estrous mare. The testosterone propionate-treated mares mounted and bit estrous mares more frequently than the stallion but exhibited less sniffing, flehmen and vocalization behavior in the presence of an estrous mare than the stallion. Testosterone propionate-treated mares and the stallion mounted an estrous mare 23.3 +/- 9.7 seconds and 172.5 +/- 22.5 seconds, respectively, after being introduced into the pen. Mares in estrus were mounted by the testosterone propionate-treated mares and the stallion an average of 4.0 +/- 1.3 and 1.0 +/- 0 times, respectively, during a ten-minute test. None of the non-estrous mares was ever mounted by the testosterone propionate-treated mares. In summary, testosterone propionate induced male sex behavior in intact mares and the testosterone propionate-treated mares effectively detected estrous mares.