Hybridization and reticulate evolution in Diphasiastrum (flat-branched clubmosses, Lycopodiaceae) - New data from the island of Taiwan and Vietnam.

In the species groups related to Diphasiastrum multispicatum and D. veitchii, hybridization was investigated in samples from northern and southern Vietnam and the island of Taiwan, including available herbarium specimens from southeast Asia. The accessions were analyzed using flow cytometry (living material only), Sanger sequencing and multiplexed inter-simple sequence repeat genotyping by sequencing. We detected two cases of ancient hybridization involving different combinations of parental species; both led via subsequent duplication to tetraploid taxa. A cross D. multispicatum × D. veitchii from Malaysia represents D. wightianum, a tetraploid taxon according to reported DNA content measurements of dried material (genome formulas MM, VV and MMVV, respectively). The second case involves D. veitchii and an unknown diploid parent (genome formula XX). Three hybridogenous taxa (genome formulas VVX, VVXX, VVVX) were discernable by a combination of flow cytometry and molecular data. Taxon I (VVX, three clones found on Taiwan island) is apparently triploid. Taxon II represents another genetically diverse and sexual tetraploid species (VVXX) and can be assigned to D. yueshanense, described from Taiwan island but occurring as well in mainland China and Vietnam. Taxon III is as well most likely tetraploid (VVVX) and represented by at least one, more likely two, clones from Taiwan island. Taxa I and III are presumably asexual and new to science. Two independently inherited nuclear markers recombine only within, not between these hybrids, pointing towards reproductive isolation. We present an evolutionary scheme which explains the origin of the hybrids and the evolution of new and fully sexual species by hybridization and subsequent allopolyploidization in flat-branched clubmosses.

Data on the occurrence of corticolous myxomycetes from Denali National Park, Alaska.

This data set contains data about corticolous (bark-inhabiting) myxomycetes from a 100×100 m(2) plot including ca. 380 trees of Picea glauca (white spruce), of which 260 were large enough that bark could been sampled to prepare moist chamber cultures. At the end of the data set records of myxomycetes from 66 moist chambers prepared with bark of deciduous trees and shrubs, and outermost twiglets of P. glauca are included. These were sampled around the plot for purposes of comparison. A second data set shows measured tree parameters for the 380 trees examined in the plot. Data were used for a statistical analysis to search for environmental factors decisive for the occurrence of corticolous myxomycetes (Schnittler et al., 2016) [1].

Quantitative morphodynamics of endothelial cells within confluent cultures in response to fluid shear stress.

To evaluate shear stress-induced effects on cultured cells we have extended the mechanical setup of a multichannel in vitro rheological system and developed software allowing entire processing control and image data analysis. The values of cell motility, degree of orientation (alignment), and cell elongation were correlated as a function of time (morphodynamics). Collective and individual endothelial cells within confluent cultures displayed a shear stress-dependent characteristic phase behavior of the following time course: resting conditions (phase I), change of motility (phase II), onset of alignment (phase III), and finally cell elongation (phase IV). Especially cell motility was characterized by a randomized zigzag movement around mean trajectories (fluctuations) together with mean cell locomotion. Onset of shear stress caused a down-regulation of fluctuations of 30% within <10 min and simultaneously increased locomotion velocities preferring the flow direction (phase II). After a lag period of 10 to 20 min cells orientated in the direction of flow (phase III) without significant cell elongation, which finally occurs within hours (phase IV). These data provide first evidence that cells within confluent endothelial monolayers respond to shear stress with a characteristic phase behavior.

Characterization of human vascular endothelial cadherin glycans.

The glycosylation pattern of human vascular endothelial cadherin (VE-cadherin), purified from cultured human umbilical cord vein endothelial cells, was analyzed. VE-cadherin was metabolically radiolabeled with d-[6-(3)H]glucosamine, isolated by immunoprecipitation, purified by SDS-PAGE and in-gel digested with endoproteinase Asp N. Oligosaccharides were sequentially released from resulting glycopeptides and analyzed by chromatographic profiling. The results revealed that VE-cadherin carries predominantly sialylated diantennary and hybrid-type glycans in addition to some triantennary and high mannose-type species. Highly branched, tetraantennary oligosaccharides were found in trace amounts only. Immunohistochemical labeling of VE-cadherin and sialic acids displayed a codistribution along the intercellular junctions in endothelial cells of human umbilical arteries, veins, and cultured endothelial monolayers. Ca(2+)-depletion, performed on cultured endothelial cells, resulted in a reversible complete disappearance of VE-cadherin and of almost all sialic acid staining from the junctions. Sialidase treatment of whole cells caused a change of VE-cadherin immunofluorescence from a continuous and netlike superstructural organization to a scattered inconsistent one. Hence, cell surface sialic acids might play a role in VE-cadherin organization.

Evidence for N-linked glycosylation in Toxoplasma gondii.

In this paper we report experiments demonstrating the presence of N-linked oligosaccharide structures in Toxoplasma gondii tachyzoites, providing the first direct biochemical evidence that this sporozoan parasite is capable of synthesizing N-linked glycans. The tachyzoite surface glycoprotein gp23 was metabolically labelled with [3H]glucosamine and [3H]mannose. Gel-filtration chromatography on Bio-Gel P4 columns produced four radiolabelled N-linked glycopeptides which were sensitive to peptidase-N-glycanase F, but resistant to endoglycosidases H and F. Using chemical analysis and exoglycosidase digestions followed by Dionex-high-pH anion-exchange chromatography and size fractionation on Bio-Gel P4 we show that gp23 has N-linked glycans in the hybrid- or complex-type structure composed of N-acetylgalactosamine, N-acetylglucosamine and mannose and devoid of sialic acid and fucose residues. In addition, the sensitivity of glycopeptides from glycoprotein extracts to endoglycosidases H and F revealed the in vivo synthesis of oligomannose-type structures by T. gondii tachyzoites. We have extended these findings by demonstrating the ability of T. gondii microsomes to synthesize in vitro a glucosylated lipid-bound high-mannose structure (Glc3Man9GlcNAc2) that is assumed to be identical with the common precursor for N-glycosylation in eukaryotes.

Apparent lack of N-glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum.

This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum, and the presence in the infected erythrocyte of the respective precursors. In in vitro cultures, P. falciparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or O-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into O-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. falciparum, using exogenous glycosyl donors and acceptors. Our studies show that O-glycosylation is the major form of protein glycosylation in intraerythrocytic P. falciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A., Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372, 645].

Binding characteristics and functional G protein coupling of muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes.

The non-selective labelled antagonist [3H]N-methyl-scopolamine ([3H]NMS) was used to identify muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes. Saturation and kinetic experiments revealed a binding site with a KD-value of 0.2-0.3 nmol/l and a receptor concentration (Bmax) of 100 fmol/mg protein. The affinities of eight selective muscarinic antagonists were determined and compared with those at M1 (rat cerebral cortex), M2 (rat heart), M3 (rat submandibular gland) and M4 (data from Dörje et al. 1991) receptors. The "M2-selective" agent AF-DX 116, the group of "M2/M4-selective" compounds himbacine, AF-DX 384, AQ-RA 741 and methoctramine but also the "M3-selective" HHSiD showed affinities corresponding to M2 and/or M4 sites. The intermediate affinity of 4-DAMP favours a mixed M2/M4 receptor population mainly containing M2 receptors. Two compounds, pirenzepine and AQ-RA 741, displayed biphasic displacement curves indicating the presence of a small population of putative M1 receptors. The rat duodenum antagonist binding profile, however, is not consistent with the presence of M3 receptors. We further demonstrate a concentration-dependent stimulation of [35S]GTP[S] binding to duodenal G proteins by the muscarinic agonist oxotremorine. Estimation of the binding parameters of GTP[S] in absence and presence of oxotremorine provided evidence for a catalytic activation of G proteins by agonist-activated muscarinic receptors in rat duodenal membranes and a strong signal amplification on the G protein level.

Structure-activity relationships of cyclic beta-casomorphin-5 analogues.

Cyclic analogues of the beta-casein-derived opioid peptide beta-casomorphin-5 (H-Tyr-Pro-Phe-Pro-Gly-OH) were prepared through substitution of the Pro2 residue with various alpha,omega-diamino acid residues (lysine, ornithine, 2,4-diaminobutyric acid) and cyclization of the omega-amino group to the C-terminal carboxyl function. Compounds of this type, with D-configuration at the 2-position residue, showed high opioid receptor affinity with some preference for mu receptors over delta receptors, high potency in the guinea pig ileum assay and considerable activity in the mouse vas deferens assay. Configurational inversion at the 4-position in these cyclic analogues resulted in enhanced affinity for both mu and delta receptors, whereas N-methylation of the Phe3 residue produced a potency decrease.

Antagonist binding reveals two heterogenous B2 bradykinin receptors in rat myometrial membranes.

In rat myometrial membranes, two bradykinin binding sites with K1 values of 18 pM and 5.6 nM were identified. Three potent bradykinin antagonists were tested for their ability to compete for [3H]bradykinin binding. Two of them, D-Arg[Hyp3,Thi5.8,D-Phe7]bradykinin and [Hyp3,Thi5.8,D-Phe7]bradykinin, also bound to both the high- (KH) and the low-affinity (KL) site whereas [Thi5.8,D-Phe7]bradykinin identified only the low-affinity bradykinin receptor. There is a close correlation between the antagonistic potencies and the KL site affinities.

Structure-activity studies of novel casomorphin analogues: binding profiles towards mu 1-, mu 2- and delta -opioid receptors.

The beta -casomorphin-5 sequence was systematically modified by substitution of the naturally occurring amino acids. The derivatives are compared on the basis of their affinities towards mu 1-, mu 2 and delta -opioid binding sites estimated by means of binding studies with [3H]dihydromorphine and [3H]D-Ala2-Leu5-enkephalin as labels and computer curve fitting. A C-terminal amide group which is known to increase mu-site affinity of beta -CM-4 and beta -CM-5 mainly enhances the mu 2-site affinity. Furthermore, the Pro4-amide structure, which seems to be responsible for the affinity enhancement can be substituted by the pyrrolidide ring structure. Modifications in position 2, 3 and 4 can lead to an increase in mu 1-, mu 2- or delta -site affinity and/or selectivity. The specificity of these effects might be dependent on the respective changes in the charge or hydrophobicity due to the introduction of other amino acids. The results suggest firstly that the alternating proline residues in the beta -CM-5 molecule are not absolutely necessary for its mu-site affinities, and secondly that both opioid receptor subtype affinity and selectivity may be modified by changing charge and/or hydrophobicity in the middle part of the beta -casomorphin molecule.

High-affinity bradykinin receptor-catalyzed G protein activation in rat myometrium.

Binding of the labelled GTP analog, guanosine-5'-O-(3-thiotriphosphate ([35S]GTP[S] to G proteins was studied in rat myometrial membranes in the presence of GDP (1 microM). Binding was stimulated by bradykinin at subnanomolar concentrations. while oxotremorine increased binding of [35S]GTP[S] to myometrial membranes at micromolar concentrations. The bradykinin-induced stimulation was antagonized by the receptor antagonist, [D-Arg-(Hyp3, Thi5,8, D-Phe7)]bradykinin. Addition of NaCl (150 nM) decreased control binding and abolished the stimulatory effect of bradykinin. On the other hand, addition of CaCl2 (5 mM) had no effect on control binding but also prevented the bradykinin-induced increased in [35S]GTP[S] binding. Saturation experiments revealed that activation of the bradykinin receptor leads to about a three-fold increase in the apparent GTP[S] binding affinity of about 30% of the total GTP[S] binding sites measured in these membranes. The results provide evidence for a high-affinity bradykinin receptor in rat myometrial membranes which interacts with and activates G proteins. This receptor action, which appears to be under the control of both sodium and calcium ions, is catalytic and leads to a large signal amplification, in that one agonist-liganded bradykinin receptor can apparently activate up to 100 G proteins.

Nonopioid effects of beta-casomorphin-5 in guinea pig heart: alterations to the beta-adrenoceptor-G-protein complex and inhibition of myocardial responses to isoproterenol.

The influence of beta-casomorphin-5 on the beta-adrenoceptor complex in guinea pig heart membranes was studied by means of binding studies, G-protein investigations and isolated heart preparations. In nanomolar concentrations beta-CM-5 induced an increase in receptor affinity towards the agonist isoproterenol whereas the antagonist affinity was reduced. The isoproterenol-stimulated increase in cardiac contractility, moreover, is reduced by 10 nM beta-CM-5. Furthermore, beta-CM-5 was found to inhibit the isoproterenol-induced GDP/GTP exchange as well as the [35S]GTP[S] binding to guinea pig heart membranes, indicating an involvement of G-proteins. These findings suggest that low concentrations of beta-CM-5 modulate the functional properties of the myocardial beta-adrenoceptor-G-protein complex, presumably resulting in its desensitization. The observed effects of beta-CM-5 are not prevented by naloxone and, therefore, are nonopioid in nature.

A high-affinity bradykinin receptor in membranes from rat myometrium is coupled to pertussis toxin-sensitive G-proteins of the Gi family.

In rat myometrial membranes, two 3H-Bradykinin binding sites with KD values of 16 pM and 1.0 nM were identified. Employed at pM concentrations, bradykinin stimulated high affinity GTPases. This effect was abolished by the bradykinin antagonist, [D-Arg(Hyp3-Thi5,8, D-Phe7)]bradykinin (10 microM), and by treatment of membranes with pertussis toxin. Myometrial membranes contained two pertussis toxin substrates of 40 and 41 kDa, which corresponded immunologically to alpha-subunits of Gi-type G-proteins. The faster migrating substrate was tentatively identified as Gi2 alpha-subunit. The electrophoretic mobility of the slower migrating Gi alpha-subunit was very similar to that of the Gi3 alpha-subunit. Go alpha-subunits were not detected. Thus, in uterine smooth muscle, G-proteins of the Gi-family (Gi2, Gi3) couple high-affinity bradykinin receptors to their effector enzymes.

[3H]naloxone as an opioid receptor label: analysis of binding site heterogeneity and use for determination of opioid affinities of casomorphin analogues.

The nonselective antagonist [3H]naloxone was used to identify opioid receptors in rat brain membranes. The multiple naloxone binding sites were related to different opioid receptors by means of selective opioid ligands as well as various beta-casomorphin analogues. Analysis of binding site heterogeneity was performed using several computer curve fitting methods. The results indicate that structurally modified casomorphin peptides are able to discriminate between mu 1- and mu 2-binding sites. The affinities to the mu-sites obtained with [3H]naloxone as label are in a good agreement with those from experiments with the mu-selective radioligand [3H]DAGO. The mu 1-site affinities of these casomorphin derivatives are well correlated with their antinociceptive potencies. This finding suggests the mediation of the analgesic activity via the high-affinity mu 1-subtype.