Heme oxidation in a chimeric protein of the alpha-selective Neisseriae meningitidis heme oxygenase with the distal helix of the delta-selective Pseudomonas aeruginosa

Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeruginosa (pa-HO) share significant sequence identity (37%). In nm-HO, biliverdin IXalpha is the sole product of the reaction, whereas pa-HO yields predominantly biliverdin IXdelta. We have previously shown by NMR that the in-plane conformation of the heme in pa-HO is significantly different from that of nm-HO as a result of distinct interactions of the heme propionates with the protein scaffold [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. In the report presented here, we have extended these studies to investigate the role of the distal helix by preparing a chimera of nm-HO (nm-HOch), in which distal helix residues 107-142 of nm-HO have been replaced with the corresponding residues of the delta-regioselective pa-HO (112-147). Electronic absorption spectra, resonance Raman and FTIR spectroscopic studies confirm that the orientation and hydrogen bonding properties of the proximal His ligand are not significantly altered in the chimera relative those of the wild-type proteins. The catalytic turnover of the nm-HOch-heme complex yields almost exclusively alpha-biliverdin and a small but reproducible amount of delta-biliverdin. NMR spectroscopic studies reveal that the altered regioselectivity in the chimeric protein likely stems from a dynamic equilibrium between two alternate in-plane conformations of the heme (in-plane heme disorder). Replacement of K16 with Ala and Met31 with Lys in the chimeric protein in an effort to tune key polypeptide-heme propionate contacts largely stabilizes the in-plane conformer conducive to delta-meso hydroxylation.     

Development of a bioassay to assess resistance to Fusarium oxysporum (Schlecht.) in asparagus (Asparagus officinalis L.)

Fusarium oxysporum is one of the major pathogens causing root and crown rot in asparagus. Breeding of cultivars resistant to F. oxysporum would be the most efficient strategy for pathogen control. In this study, a bioassay was developed for screening seedling resistance. The non‐destructive bioassay comprises inoculation with a highly aggressive F. oxysporum isolate, incubation in a climate chamber and quantification of disease symptoms by a digital image analysing system and a PTA‐ELISA. This bioassay is simple to implement and demonstrated high reproducibility. Subsequently, it was used to determine the resistance behaviour of 16 asparagus genotypes to F. oxysporum. The asparagus cultivars revealed different levels of susceptibility, whereas the wild relative A. densiflorus was confirmed to be resistant.     

rbcL sequences support exclusion ofRetzia,Desfontainia, andNicodemia from theGentianales

The taxonomic positions ofRetzia, Desfontainia, andNicodemia have been much discussed, and all three genera have been included inLoganiaceae (Gentianales). We have made a cladistic analysis ofrbcL gene sequences to determine the relationships of these taxa toGentianales. Four newrbcL sequences are presented; i.e., ofRetzia, Desfontainia, Diervilla (Caprifoliaceae), andEuthystachys (Stilbaceae). Our results show thatRetzia, Desfontainia, andNicodemia are not closely related toLoganiaceae or theGentianales. Retzia is most closely related toEuthystachys and is better included inStilbaceae. The positions ofDesfontainia andNicodemia are not settled, butDesfontainia shows affinity for theDipsacales s.l. andNicodemia for theLamiales s.l.     

Net taurine transport and its inhibition by a taurine antagonist

P₂-fractions were isolated from rat brain, and used to study net taurine transport. The fractions were incubated in increasing concentrations of [³H]taurine and the intraterminal concentration measured by liquid scintillation and amino acid analysis. The membrane potential of the isolated fractions was estimated using⁸⁶Rb⁺ as a marker for intracellular K⁺. Taurine was synthesized in the P₂-fraction when incubated in taurine free medium. At external taurine concentrations below 370 M a significant amount of the endogenous taurine was released to the incubation medium. Net taurine uptake into the P₂-fraction was achieved at external taurine concentrations exceeding 370 M. The taurine antagonist 6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide (TAG) competitively inhibited taurine and [³H]taurine transport into the P₂-fraction. As the external concentration of taurine was increased, the accumulation of⁸⁶Rb⁺ into the P₂-fraction was facilitated. This indicated an increasing hyperpolarization of the neuronal membrane as taurine transport shifted from release towards uptake. TAG reduced the hyperpolarization that paralleled taurine accumulation, in a dose dependent manner. Our results indicate that relatively low transmembranal gradients of taurine may be maintained by an electrogenic taurine transporter having a large transport capacity. Such a transporter may well serve the needs of osmotic regulation, i.e. to transport large amounts of taurine in any direction across the neuronal membrane.     

Microbial processes in a high-latitude fjord (Kongsfjorden, Svalbard): II. Ciliates and dinoflagellates

The composition and ecological role of ciliates and dinoflagellates were investigated at one station in Kongsfjorden, Svalbard, during six consecutive field campaigns between March and December 2006. Total ciliate and dinoflagellate abundance mirrored the seasonal progression of phytoplankton, peaking with 5.8 × 10⁴ cells l⁻¹ in April at an average chlorophyll a concentration of 10 μg l⁻¹. Dinoflagellates were more abundant than ciliates, dominated by small athecates. Among ciliates, aloricate oligotrichs dominated the assemblage. A large fraction (>60%) of ciliates and dinoflagellates contained chloroplasts in spring and summer. The biomass of the purely heterotrophic fraction of the ciliate and dinoflagellate community (protozooplankton) was with 14 μg C l⁻¹ highest in conjunction with the phytoplankton spring bloom in April. Growth experiments revealed similar specific growth rates for heterotrophic ciliates and dinoflagellates (<0–0.8 d⁻¹). Food availability may have controlled the protozooplankton assemblage in winter, while copepods may have exerted a strong control during the post-bloom period. Calculations of the potential grazing rates of the protozooplankton indicated its ability to control or heavily impact the phytoplankton stocks at most times. The results show that ciliates and dinoflagellates were an important component of the pelagic food web in Kongsfjorden and need to be taken into account when discussing the fate of phytoplankton and biogeochemical cycling in Arctic marine ecosystems.     

Surface structure and anatomical aspects of Chinese brake fern (Pteris vittata; Pteridaceae)

The objective of this study was to describe a wide spectrum of surface structural and anatomical details of the Chinese brake fern (Pteris vittata) using scanning electron microscopy (SEM). SEM revealed that the epidermal cells of the pinnae were elongated with raised periclinal and sinuous anticlinal walls. The pinnae were hypostomatous with randomly scattered anomocytic stomatal complexes positioned at the same level as the epidermis. Stomates were large and elliptical (27.4 μm × 10.2 μm). Cross sections from the central regions of the rachis and the stipe revealed V- and U-shaped vascular bundles, respectively. In each vascular bundle, the xylem strands were sea-horse shaped (hippocampus). In contrast, the pinnae possessed a triangular vascular bundle with uniform mesophyll organization comprising of homogenous lobed parenchyma cells. The indumentum consisted of trichomes and scales, which formed various types of vestiture. Trichomes were borne only on the pinnae and scales on the rachis and stipe. The roots developed a dense network of long root hairs averaging 244 μm long, and the xylem consisted of tracheids with scalariform pitting. Sori were submarginal; continuous along both margins of the pinna and were covered with a false indusium. The sporangia were oblong with a short thick stalk and the annulus was positioned vertically resulting in transverse dehiscence of the sporangium. The paraphyses were uniseriate, unbranched, septate and found to be intermixed with the sporangia. The exine of the globose spores was adorned with thick reticulum in which the areoles contained round tubercles. This study describes surface features in detail, which is essential to studies examining the issue of whether morphological characteristics are related to arsenic hyperaccumulation inP. vittata.     

Mobbing behaviour in a passerine community increases with prevalence in predator diet

Mobbing behaviour against predators is well documented but less is known about the factors influencing variation in behavioural response between prey species. We conducted a series of playback experiments to examine how the mobbing responses of prey species differed according to their relative risk of predation by the Eurasian Pygmy Owl Glaucidium passerinum, a predator of passerines. We found that mobbing among 22 passerine prey species was positively correlated with their prevalence in the Pygmy Owl diet. To compare mobbing behaviour between two seasons, we conducted playback experiments during spring (breeding season) and autumn (non‐breeding season). Contrary to previous studies, we found that mobbing intensity was greater during autumn than in spring. Our study shows a differential mobbing response of 22 species to the calls from one predator species and underscores the importance of considering seasonal variation in mobbing behaviour. Mobbing response differences observed among bird species strongly suggest different cooperation behaviour at the community level.     

Glucagon stimulates exocytosis in mouse and rat pancreatic alpha-cells by binding to glucagon receptors

Glucagon, secreted by the pancreatic alpha-cells, stimulates insulin secretion from neighboring beta-cells by cAMP- and protein kinase A (PKA)-dependent mechanisms, but it is not known whether glucagon also modulates its own secretion. We have addressed this issue by combining recordings of membrane capacitance (to monitor exocytosis) in individual alpha-cells with biochemical assays of glucagon secretion and cAMP content in intact pancreatic islets, as well as analyses of glucagon receptor expression in pure alpha-cell fractions by RT-PCR. Glucagon stimulated cAMP generation and exocytosis dose dependently with an EC50 of 1.6-1.7 nm. The stimulation of both parameters plateaued at concentrations beyond 10 nm of glucagon where a more than 3-fold enhancement was observed. The actions of glucagon were unaffected by the GLP-1 receptor antagonist exendin-(9-39) but abolished by des-His1-[Glu9]-glucagon-amide, a specific blocker of the glucagon receptor. The effects of glucagon on alpha-cell exocytosis were mimicked by forskolin and the stimulatory actions of glucagon and forskolin on exocytosis were both reproduced by intracellular application of 0.1 mm cAMP. cAMP-potentiated exocytosis involved both PKA-dependent and -independent (resistant to Rp-cAMPS, an Rp-isomer of cAMP) mechanisms. The presence of the cAMP-binding protein cAMP-guanidine nucleotide exchange factor II in alpha-cells was documented by a combination of immunocytochemistry and RT-PCR and 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP, a cAMP-guanidine nucleotide exchange factor II-selective agonist, mimicked the effect of cAMP and augmented rapid exocytosis in a PKA-independent manner. We conclude that glucagon released from the alpha-cells, in addition to its well-documented systemic effects and paracrine actions within the islet, also represents an autocrine regulator of alpha-cell function.