Ultrastructure and development during meiosis and the tetrad period of sporogenesis in the leptosporangiate fern Alsophila setosa (Cyatheaceae) compared with corresponding stages in Psilotum nudum (Psilotaceae)

The pre-meiotic, meiotic and tetrad stages of development in microsporangia of Alsophila setosa were studied with particular emphasis on the early establishment of patterning in the microspore wall and the subsequent development of the sporoderm. The data obtained were compared with corresponding ontogenetic stages of Psilotum nudum. Tapetal behaviour was also examined. During the tetrad period, only one layer, a thin undulating sheet, appeared alongside the plasma membrane of the tetraspores, and this was evidently formed on a pre-patterned structure – a fibrillar layer, corresponding to a kind of primexine matrix. The early free microspores had a wavy plasma membrane with a parallel, sinusoidal, thin initial sporoderm layer. The proximal apertural fold was observed to be an extended outgrowth of this initial spore envelope. Sporoderm ontogeny during the tetrad period in Alsophila and Psilotum show some common points, but also fundamental differences, mainly in the relative timing of events: in Alsophila the end of the tetrad period is the starting point for exospore development, whereas in Psilotum the exospore is already complete at this stage. Considerable differences were also observed in the tapetum of the two species.     

Design,synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: Observation of extensive dynamics in an enzyme/inhibitor complex

The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH₂ group, inhibits HDHD4 with a binding affinity (IC₅₀ 11 μM) in the range of the native substrate Neu5Ac-9-P (compound 1, K_m 47 μM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg²⁺. In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca²⁺ and Mg²⁺ are indicative of a highly dynamic process in the active site for the HDHD4/Mg²⁺/3 complex. Possible explanations for this observation are discussed.     

Development of a pharmacophore model for the catecholamine release-inhibitory peptide catestatin: Virtual screening and functional testing identify novel small molecule therapeutics of hypertension

The endogenous catecholamine release-inhibitory peptide catestatin (CST) regulates events leading to hypertension and cardiovascular disease. Earlier we studied the structure of CST by NMR, molecular modeling, and amino acid scanning mutagenesis. That structure has now been exploited for elucidation of interface pharmacophores that mediate binding of CST to its target, with consequent secretory inhibition. Designed pharmacophore models allowed screening of 3D structural domains. Selected compounds were tested on both cultured catecholaminergic cells and an in vivo model of hypertension; in each case, the candidates showed substantial mimicry of native CST actions, with preserved or enhanced potency and specificity. The approach and compounds have thus enabled rational design of novel drug candidates for treatment of hypertension or autonomic dysfunction.     

Luteibacter rhizovicinus MIMR1 promotes root development in barley (Hordeum vulgare L.) under laboratory conditions

In order to preserve environmental quality, alternative strategies to chemical-intensive agriculture are strongly needed. In this study, we characterized in vitro the potential plant growth promoting (PGP) properties of a gamma-proteobacterium, named MIMR1, originally isolated from apple shoots in micropropagation. The analysis of the 16S rRNA gene sequence allowed the taxonomic identification of MIMR1 as Luteibacter rhizovicinus. The PGP properties of MIMR1 were compared to Pseudomonas chlororaphis subsp. aurantiaca DSM 19603^T, which was selected as a reference PGP bacterium. By means of in vitro experiments, we showed that L. rhizovicinus MIMR1 and P. chlororaphis DSM 19603^T have the ability to produce molecules able to chelate ferric ions and solubilize monocalcium phosphate. On the contrary, both strains were apparently unable to solubilize tricalcium phosphate. Furthermore, the ability to produce 3-indol acetic acid by MIMR1 was approximately three times higher than that of DSM 19603^T. By using fluorescent recombinants of strains MIMR1 and DSM 19603^T, we also demonstrated that both bacteria are able to abundantly proliferate and colonize the barley rhizosphere, preferentially localizing on root tips and in the rhizoplane. Finally, we observed a negative effect of DSM 19603^T on barley seed germination and plant growth, whereas MIMR1, compared to the control, determined a significant increase of the weight of aerial part (+22 %), and the weight and length of roots (+53 and +32 %, respectively). The results obtained in this work make L. rhizovicinus MIMR1 a good candidate for possible use in the formulation of bio-fertilizers.     

Atrioventricular nodal function during atrial fibrillation: Model building and robust estimation

Statistical modeling of atrioventricular (AV) nodal function during atrial fibrillation (AF) is revisited for the purpose of defining model properties and improving parameter estimation. The characterization of AV nodal pathways is made more detailed and the number of pathways is now determined by the Bayesian information criterion, rather than just producing a probability as was previously done. Robust estimation of the shorter refractory period (i.e., of the slow pathway) is accomplished by a Hough-based technique which is applied to a Poincaré plot of RR intervals. The performance is evaluated on simulated data as well as on ECG data acquired from AF patients during rest and head-up tilt test. The simulation results suggest that the refractory period of the slow pathway can be accurately estimated even in the presence of many artifacts. They also show that the number of pathways can be accurately determined. The results from ECG data show that the refined AV node model provides significantly better fit than did the original model, increasing from 85 ± 5% to 88 ± 4% during rest, and from 86 ± 5% to 87 ± 3% during tilt. When assessing the effect of sympathetic stimulation, the AF frequency increased significantly during tilt (6.25 ± 0.58 Hz vs. 6.32 ± 0.61 Hz, p < 0.05, rest vs. tilt) and the prolongation of the refractory periods of both pathways decreased significantly (slow pathway: 0.23 ± 0.20 s vs. 0.11 ± 0.10 s, p < 0.001, rest vs. tilt; fast pathway: 0.24 ± 0.31 s vs. 0.16 ± 0.19 s, p < 0.05, rest vs. tilt). The results show that AV node characteristics can be assessed noninvasively for the purpose of quantifying changes induced by autonomic stimulation.     

Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site‐directed mutagenesis

Multicopper oxidases can act on a broad spectrum of phenolic and non‐phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid), 2,6‐dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where k_cat and K_m values at 60°C were 8.1 (± 0.8) s^?1 and 0.9 (± 0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH 4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half‐life of over 10 h at 80°C and over 7 h at 90°C. Site‐directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10‐fold increase in activity compared with the wild‐type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta‐position of aromatic substrates.     

Protection of Cells against Oxidative Stress by Nanomolar Levels of Hydroxyflavones Indicates a New Type of Intracellular Antioxidant Mechanism

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.     

Reduced Crowding and Poor Contour Detection in Schizophrenia Are Consistent with Weak Surround Inhibition

Background

Detection of visual contours (strings of small oriented elements) is markedly poor in schizophrenia. This has previously been attributed to an inability to group local information across space into a global percept. Here, we show that this failure actually originates from a combination of poor encoding of local orientation and abnormal processing of visual context.

Methods

We measured the ability of observers with schizophrenia to localise contours embedded in backgrounds of differently oriented elements (either randomly oriented, near-parallel or near-perpendicular to the contour). In addition, we measured patients’ ability to process local orientation information (i.e., report the orientation of an individual element) for both isolated and crowded elements (i.e., presented with nearby distractors).

Results

While patients are poor at detecting contours amongst randomly oriented elements, they are proportionally less disrupted (compared to unaffected controls) when contour and surrounding elements have similar orientations (near-parallel condition). In addition, patients are poor at reporting the orientation of an individual element but, again, are less prone to interference from nearby distractors, a phenomenon known as visual crowding.

Conclusions

We suggest that patients’ poor performance at contour perception arises not as a consequence of an “integration deficit” but from a combination of reduced sensitivity to local orientation and abnormalities in contextual processing. We propose that this is a consequence of abnormal gain control, a phenomenon that has been implicated in orientation-selectivity as well as surround suppression.