Through-bond correlation of adenine H2 and H8 protons in unlabeled DNA fragments by HMBC spectroscopy

Summary A new application of the HMBC experiment is presented that provides a useful means to discriminate between H2 and H8 proton resonances, to assign the base proton resonances to the various residue types and, most importantly, to correlate the H2 and H8 protons for adenine or inosine residues in natural abundance ¹³C fragments. The utility of this experiment is demonstrated for an unlabeled DNA 20-mer. Thanks to the obtained results, preliminary conclusions could be drawn regarding the molecular conformations of the non-canonical G/I-A base pairs in the hairpin formed by this fragment.     

Transpiration and drought resistance of Douglas-fir seedlings exposed to excess ammonium

 In a pot trial growth and transpiration of 3-year-old Douglas-fir seedlings on an acid, sandy soil was examined at a deficient (30 kg N ha^{–  1} year^{–  1}) and an excessive level (120 kg N ha^{–  1} year^{–  1}) of NH₄ application. Dissolved ammonium sulphate was applied to the pots weekly for two growing seasons. In half of the pots a complete set of other nutrients was applied in optimal proportions to the applied nitrogen. Water supply was optimal and transpiration was recorded. At the end of the second treatment season irrigation was stopped for 2 weeks during dry and sunny weather. Both high application of NH₄ and additional nutrients increased shoot growth and transpiration demand in the first treatment year. The root system was smaller at higher N level and this reduced water uptake accordingly. In the second year the combination of high NH₄ ⁺ and additional nutrients affected root functioning predominantly due to salinity effects and this seriously decreased water uptake capacity and shoot water potentials, finally resulting in tree death. Without addition of other nutrients the high NH₄ ⁺ application resulted in a high degree of soil acidification, which damaged the roots, that showed a decrease in water uptake capacity. At the low NH₄ supply level soil acidification was lower, and root functioning was not affected, and the trees recovered quickly from the imposed drought. Higher needle K and P status depressed transpiration rates at the low NH₄ application rate. Received: 9 January 1995 / Accepted: 18 September 1995     

The effect of light levels on daily patterns of chlorophyll fluorescence and organic acid accumulation in the tropical CAM treeClusia hilariana

Sandy plains are characteristic of the coastal region of Brazil. We investigated the diel patterns of changes in organic acid levels, leaf conductance and chlorophylla fluorescence for sun-exposed and shaded plants ofClusia hilariana, one of the dominant woody species in the sandy coastal plains of northern Rio de Janeiro state. Both exposed and shaded plants showed a typical CAM pattern with considerable diel oscillations in organic acid levels. The degradation of both malic and citric acids during the midday stomatal closure period could lead to potential CO₂ fixation rates of 28 mol m^-2 s^-1 in exposed leaves. Moreover, exposed leaves exhibited large increases in total non-photochemical quenching (q_N) accompanied by a substantial decrease in effective quantum yield during the course of the day. However, these potential high rates of CO₂ fixation and the increases inqn of exposed plants were not enough to maintain the primary electron acceptor of photosystem II (q_A) in a low reduction state, similar to that of shaded plants. As a result, there was a moderate increase in the reduction state of q_A throughout the day. Most of the decline in photochemical efficiency of exposed leaves ofC. hilariana was reversible, as evidenced by the high levels of pre-dawn potential quantum yields (F_v/F_m) and their rapid recovery after sunset. However, the depletion of the organic acid pool in the afternoon resulted in an accentuated subsequent drop in F_v/F_m, suggesting that prolonged periods of water stress accompanied by high irradiance levels may expose plants ofC. hilariana in unprotected habitats to the danger of photoinhibition.     

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

Structural and immunochemical studies on the lipopolysaccharide of the ‘T-antigen’-containing mutant Proteus mirabilis R14/1959

Abstract In DOC-PAGE, lipopolysaccharide (LPS) of Proteus mirabilis R14/1959 (Rb-type) mutant showed a ladder-like migration pattern indicating the presence of a high molecular weight polysaccharide chain. The isolated polysaccharide, called T-antigen because of similarity with the T1 chain of Salmonella friedenau LPS, contained d -glucose, d -galacturonic acid ( d -GalA), and d -GlcNAc in molar ratios 2:1:1 and was structurally different from the O-antigen of the parental S-strain P. mirabilis S1959 but identical to the O-antigen of another S-strain Proteus penneri 42. The importance of a d -GalA( l -Lys)-containing epitope, most likely present in the core region of LPS, and of GalA present in the T-antigen chain in manifesting the serological specificity of P. mirabilis R14/1959 were revealed using rabbit polyclonal homologous and heterologous R- and O-specific antisera and the appropriate antigens, including synthetic antigens which represent partial structures of various Proteus LPS.     

Synthesis and biological activity of tachykinin analogs containing the adamantane moiety

Summary The adamantane moiety was introduced in the tachykinin NK₂ receptor-selective agonist [-Ala⁸]-NKA(4–10) (H-Asp-Ser-Phe-Val--Ala-Leu-Met-NH₂, MEN 10210) and in different positions of the NK₂ receptor antagonist MEN 10376 (H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂) in order to investigate how this substitution affects their biological activity at tachykinin NK₁, NK₂ and NK₃ receptors. 1-Adamantaneacetic acid (1-Ada-CH₂COOH) was directly conjugated in the solid phase as the preformed OBt active ester to the N-terminal position of MEN 10210, obtaining MEN 10586 (1-Ada-CH₂CO-Asp-Ser-Phe-Val--Ala-Leu-Met-NH₂). The Pfp ester of adamantaneacetic acid (1) was prepared and used for the acylation of the N-terminal position of MEN 10376, yielding MEN 10606 (1-Ada-CH₂CO-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂). Compound 1 was then used to obtain the building block Fmoc-Lys(1-Ada-CH₂CO)-OH as a modified amino acid for the synthesis of MEN 10818 [H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys(1-Ada-CH₂CO)-NH₂]. In order to investigate the biological activity of the peptide bearing the adamantane group together with the free N-terminal amino function, we synthesised MEN 10676 [H-Asp(O-2-Ada)-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂] using Fmoc-Asp(O-2-Ada)-OH, in which 2-adamantanole was the protecting group of the aspartate -COOH moiety during the peptide synthesis and survived the final peptide cleavage and deprotection carried out under controlled conditions. MEN 10586 showed an agonist activity comparable to that of the parent compound MEN 10210 at NK₁ and NK₂ receptors of guinea pig ileum, rabbit isolated pulmonary artery and hamster isolated trachea preparations, while it showed a 25-fold higher agonist activity at NK₃ receptors of rat isolated portal vein. The three modified antagonist analogs displayed similar or reduced affinity at NK₁, NK₂ and NK₃ receptors as compared to MEN 10376. The drop was particularly evident (>2 log units) at the NK₂ receptors of the rabbit isolated pulmonay artery.     

Bat species diversity in a lake archipelago in central Sweden

The bat fauna of 35 islands in a large lake in central Sweden were examined using ultrasound detectors. We tested the hypothesis that there is no difference in species number between the mainland and the island fauna. Eight species were found. Species numbers were analysed against island area, area of some habitats (coniferous forest, deciduous forest, semi-open habitats and open habitats), degree of isolation (distance from mainland and from stepping stones) and time spent searching for bats. Species number increased with area of deciduous forest. Presence of houses tended to increase species number. There seems to be a negative relationship between species number and degree of isolation (nearly significant). The results suggest that at least three species, Myotis brandti (Eversmann, 1845), M. mystacinus (Kuhl, 1819) and Plecotus auritus (Linnaeus, 1758), are negatively affected by forest patchiness. These species occurred mainly on large islands. Thus, the results do not support the hypothesis. The reasons why some species avoid open habitats are discussed.     

Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway.

A 5-kD plant defensin was purified from Arabidopsis leaves challenged with the fungus Alternaria brassicicola and shown to possess antifungal properties in vitro. The corresponding plant defensin gene was induced after treatment of leaves with methyl jasmonate or ethylene but not with salicylic acid or 2,6-dichloroisonicotinic acid. When challenged with A. brassicicola, the levels of the plant defensin protein and mRNA rose both in inoculated leaves and in nontreated leaves of inoculated plants (systemic leaves). These events coincided with an increase in the endogenous jasmonic acid content of both types of leaves. Systemic pathogen-induced expression of the plant defensin gene was unaffected in Arabidopsis transformants (nahG) or mutants (npr1 and cpr1) affected in the salicylic acid response but was strongly reduced in the Arabidopsis mutants eln2 and col1 that are blocked in their response to ethylene and methyl jasmonate, respectively. Our results indicate that systemic pathogen-induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response.     

Relationships among didelphid marsupials based on sequence variation in the mitochondrial cytochrome B gene

Variation in the mitochondrial cytochrome b gene (nucleotide and amino acid sequences) is evaluated for 9 genera and 15 species of American opossums in the family Didelphidae, using the American caenolestid rat opossumLestoros and the New Guinean peroryctid bandicootEchimypera as outgroups. Phylogenetic analyses (parsimony and distance) strongly support the monophyly of the Didelphidae and delineate two major clades; (1)Didelphis andPhilander are strongly aligned sister taxa, withMetachirus weakly but consistently associated with them, and (2)Marmosa plusMicoureus, withMonodelphis falling outside that pair. The generaMarmosops, Caluromys, andGlironia exhibit varied relationships, depending upon the method of analysis and data (DNA or amino acid sequences) used, but generally are placed individually or in combinations near or at the base of the didelphid radiation. Some aspects of these relationships are consistent with current taxonomic views, but others are in marked contrast. Specifically, a clade comprised of the mouse opossumsMarmosa, Micoureus, andMarmosops is strongly rejected by log-likelihood analysis, contrary to expectations from some current classifications. Also, the woolly opossumsCaluromys andGlironia also do not form a sister-taxon relationship, as suggested by their placement in a subfamily separate from the remaining didelphids examined. However, such a relationship cannot be rejected from log-likelihood analyses. The relationships suggested fromcyt-b sequences are strongly concordant with those based on DNA-DNA hybridization analyses. In addition to systematic and phylogenetic properties, molecular evolution of the didelphid cytochrome b gene sequence is characterized according to nucleotide bias and rate differentials at each codon position and across the entire sequence.To whom correspondence should be addressed.