Mecardonia tenella (Plantaginaceae) Attracts Oil-, Perfume-, and Pollen-Gathering Bees in Southern Brazil

Floral characteristics often indicate the pollinators' functional group visiting the plant and the pollination syndromes associated with them. This idea has been challenged in the past decades due to increasing evidence that most plants, including those exhibiting floral syndromes, are visited by large arrays of species that differ in their effectiveness as pollinators. Our study focuses on Mecardonia tenella (Plantaginaceae) from the Araucaria forest of southern Brazil, which exhibits characteristics of the oil flower pollination syndrome. However, it is visited by three types of functional groups of bees: male orchid bees, oil-collecting bees, and pollen-seeking bees. The relative contribution of each functional group to the plant's reproductive success was estimated based on their pollen load, visitation frequency, and morphology. We assessed resources, phenology, and breeding system of M. tenella . Our results indicate that flowers lack nectar, but volatiles, lipids, and pollen are resources that can be gathered by visitors. This combination of floral traits and visitors' assemblage makes M. tenella a challenge to the concept of pollination syndromes. Our findings indicate that the current interactions may not reflect the circumstances under which some floral traits of this plant were selected.     

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

Summary The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to -galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229 Ile) and Val274 (Asp274 Val) substitutions over the N-terminal His4l (Arg4l His) substitution, and the intraallelic dominance of Thr45 (Arg45 Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88 Leu) and Arg256 (Pro256 Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Va1288 (Asp288 Val), and the double mutant was susceptible to activation by benzoates. The results suggest that intramolecular interactions between the C- and N-terminal regions of XylS are critical for activation of the regulator by the effector.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

In Vitro and In Vivo Invasiveness of Different Pulsed-Field Gel Electrophoresis Types of Listeria monocytogenes

The virulence of different pulsed-field gel electrophoresis (PFGE) types of Listeria monocytogenes was examined by monitoring their ability to invade Caco-2 cells. Strains belonging to seven different PFGE types originating from both foods and humans were included. No significant differences in invasiveness were detected between strains isolated from humans and those isolated from food. Strains belonging to PFGE type 1 expressed a significantly lower ability to invade cells compared to strains belonging to other PFGE types. Although strains of PFGE type 2 also seemed to invade at a low level, this was not significant in the present study. PFGE types 1 and 2 as well as type 14 are more frequently found in food than the four other PFGE types examined and moreover have a relatively low prevalence in humans compared to their prevalence in food. Thus, the hypothesis that some PFGE types are less virulent than others is supported by this study showing that certain PFGE types of L. monocytogenes commonly found in food are less invasive than others to Caco-2 cells. In contrast to the differences in invasion, identical intracellular growth rates between the different PFGE types were observed. In vivo studies of the actual ability of the strains to invade the liver and spleen of cimetidine-treated rats following an oral dose of 10⁹ L. monocytogenes cells were performed for isolates of PFGE types 1, 2, 5, and 15. After 2 days, equal amounts of bacteria were observed in the liver and spleen of the rats for any of the PFGE types tested.     

Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3

A lantibiotic gene cluster was identified in Bacillus subtilis A1/3 showing a high degree of homology to the subtilin gene cluster and occupying the same genetic locus as the spa genes in B. subtilis ATCC 6633. The gene cluster exhibits diversity with respect to duplication of two subtilin-like genes which are separated by a sequence similar to a portion of a lanC gene. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses of B. subtilis A1/3 culture extracts confirmed the presence of two lantibiotic-like peptides, ericin S (3,442 Da) and ericin A (2,986 Da). Disruption of the lanB-homologous gene eriB resulted in loss of production of both peptides, demonstrating that they are processed in an eriB-dependent manner. Although precursors of ericins S and A show only 75% of identity, the matured lantibiotic-like peptides reveal highly similar physical properties; separation was only achieved after multistep, reversed-phase high-performance liquid chromatography. Based on Edman and peptidase degradation in combination with MALDI-TOF MS, for ericin S a subtilin-like, lanthionine-bridging pattern is supposed. For ericin A two C-terminal rings are different from the lanthionine pattern of subtilin. Due to only four amino acid exchanges, ericin S and subtilin revealed similar antibiotic activities as well as similar properties in response to heat and protease treatment. For ericin A only minor antibiotic activity was found.     

Intervesicle cross-linking with integrin alpha IIb beta 3 and cyclic-RGD-lipopeptide. A model of cell-adhesion processes

We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer of optimal length between the peptide and lipid moieties was searched for by evaluating the binding strength with an enzyme-coupled immunosorbent assay (ELISA) and by surface plasmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the ligands, which consisted either of epsilon-aminohexanoic acid (epsilonAhx) or of epsilonAhx with two additional glycine units. Best results were obtained with c[Arg-Gly-Asp-D-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constants of K(D) = 0.158 microM from ELISA and K(D) = 1.1 microM from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-D-Phe-Lys([dimyristoyl-3-thioglyceryl-succinimido -propanoyl]Ahx-Gly-Gly)-Gly], was used as a membrane-anchored integrin ligand. It is shown by fluorescence microscopy and cryo electron microscopy that integrin reconstituted into phospholipid vesicles binds to vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allows establishment of new model systems for systematic studies of the self-organization of integrin clusters and focal adhesion complexes.