Participation of cholinergic pathways in α-hederin-induced contraction of rat isolated stomach strips

The dry extract of Hedra helix leaves and its main active compounds, predominantly α-hederin and hederacoside C, has been traditionally believed to act spasmolytic. However, it has been recently proved that both, the extract of ivy and triterpenoid saponins, exhibit strong contractile effect on rat isolated stomach smooth muscle strips. It turned out that the most potent contractile agent isolated from the extract of ivy leaves is α-hederin. Thus, it seems reasonable to estimate the mechanism of the contractile effect of this saponin. The presented study was aimed at verifying the participation of cholinergic pathways (muscarinic and nicotine receptors) in α-hederin-induced contraction. The experiments were carried out on rat isolated stomach corpus and fundus strips under isotonic conditions. The preparations were preincubated with either atropine or hexamethonium and then exposed to α-hederin. All results are expressed as the percentage of the response to acetylcholine - a reference contractile agent. The obtained results revealed that the pretreatment of isolated stomach strips (corpus and fundus) with atropine neither prevented nor remarkably reduced the reaction of the preparations to α-hederin. Similarly, if the application of saponin was preceded by the administration of hexamethonium, the strength of the contraction of stomach fundus strips induced by α-hederin was not modified. Concluding, it can be assumed that the cholinergic pathways do not participate in α-hederin-evoked contraction of rat isolated stomach preparations.     

Acetohydroxyacid synthase: a proposed structure for regulatory subunits supported by evidence from mutagenesis

Valine inhibition of acetohydroxyacid synthase (AHAS) plays an important role in regulation of biosynthesis of branched-chain amino acids in bacteria. Bacterial AHASs are composed of separate catalytic and regulatory subunits; while the catalytic subunits appear to be homologous with several other thiamin diphosphate-dependent enzymes, there has been no model for the structure of the small, regulatory subunits (SSUs). AHAS III is one of three isozymes in Escherichia coli. Its large subunit (encoded by ilvI) by itself has 3-5 % activity of the holoenzyme and is not sensitive to inhibition by valine. The SSU (encoded by ilvH) associates with the large subunit and is required for full catalytic activity and valine sensitivity. The isolated SSU binds valine. The properties of several mutant SSUs shed light on the relation between their structure and regulatory function. Three mutant SSUs were obtained from spontaneous Val(R) bacterial mutants and three more were designed on the basis of an alignment of SSU sequences from valine-sensitive and resistant isozymes, or consideration of the molecular model developed here. Mutant SSUs N11A, G14D, N29H and A36V, when reconstituted with wild-type large subunit, lead to a holoenzyme with drastically reduced valine sensitivity, but with a specific activity similar to that of the wild-type. The isolated G14D and N29H subunits do not bind valine. Mutant Q59L leads to a valine-sensitive holoenzyme and isolated Q59L binds valine. T34I has an intermediate valine sensitivity. The effects of mutations on the affinity of the large subunits for SSUs also vary. D. Fischer's hybrid fold prediction method suggested a fold similarity between the N terminus of the ilvH product and the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. On the basis of this prediction, together with the properties of the mutants, a model for the structure of the AHAS SSUs and the location of the valine-binding sites can be proposed.     

Mandibular movement patterns relative to food types in common tree shrews (Tupaia glis)

Although common tree shrews have long been considered a model system for early eutherian mastication, little information on mandibular movement patterns relative to specific food types has been reported. Detailed analysis of mandibular movement patterns when related to resulting attrition facets may permit more accurate extrapolations regarding the dietary habits of primitive mammals. Marker beads were sewn to chins of five animals that were placed in a restraint system and filmed while they fully masticated mealworm larvae and standardized pieces of banana, almond, and commercial cat chow. These sequences were divided into early, middle, and late thirds of food reduction. Mandibular positions from both frontal and lateral perspectives were digitized frame by frame to yield plots of orbits in three dimensions as well as graphic display of displacements, velocities, and accelerations. Plot coordinates were averaged to generate composite orbital shapes. Significant (p < 0.01) findings included: (1) shortest orbital durations and greatest peak closing velocities and accelerations in early third of reduction; (2) smallest maximum gape, smallest maximum lateral excursion from midline, and longest duration of powerstroke relative to orbital duration in late third of reduction; (3) shortest orbital durations and smallest maximum gape during mastication of chow; (4) greatest maximum lateral excursion during mastication of mealworm larvae; and (5) smallest peak closing accelerations during mastication of banana. Significant differences were also found among subjects for all parameters examined. Capacity for complex jaw movement may have been critical for allowing primitive molars to be used for trituration of a variety of food types, and may have preceded evolution of more specialized molar forms.     

Release of molybdenum co-factor from nitrate reductase and xanthine oxidase by heat treatment

Heat treatment (90 sec at 70°) is shown to convert the bound molybdenum co-factor of tobacco cell-free extracts and bovine milk xanthine oxidase into a form capable of complementing the Neurospora crassa mutant nit-1.In the presence of 1 mM ascorbic acid, 25 mM molybdate and, for plant extracts, sulphydryl group protecting agents, the molybdenum co-factor can survive incubations up to 100° whilst maintaining its biological activity. Especially with plant extracts, the efficiency of heat treatment is considerably higher than that of the acidification procedure which is often utilized for releasing molybdenum co-factor.     

Structure and catalytic mechanism of a novel N-succinyl-L-ornithine transcarbamylase in arginine biosynthesis of Bacteroides fragilis

A Bacteroides fragilis gene (argF'(bf)), the disruption of which renders the bacterium auxotrophic for arginine, was expressed and its recombinant protein purified and studied. The novel protein catalyzes the carbamylation of N-succinyl-L-ornithine but not L-ornithine or N-acetyl-L-ornithine, forming N-succinyl-L-citrulline. Crystal structures of this novel transcarbamylase complexed with carbamyl phosphate and N-succinyl-L-norvaline, as well as sulfate and N-succinyl-L-norvaline have been determined and refined to 2.9 and 2.8 A resolution, respectively. They provide structural evidence that this protein is a novel N-succinyl-L-ornithine transcarbamylase. The data provided herein suggest that B. fragilis uses N-succinyl-L-ornithine rather than N-acetyl-L-ornithine for de novo arginine biosynthesis and therefore that this pathway in Bacteroides is different from the canonical arginine biosynthetic pathway of most organisms. Comparison of the structures of the new protein with those recently reported for N-acetyl-L-ornithine transcarbamylase indicates that amino acid residue 90 (B. fragilis numbering) plays an important role in conferring substrate specificity for N-succinyl-L-ornithine versus N-acetyl-L-ornithine. Movement of the 120 loop upon substrate binding occurs in N-succinyl-L-ornithine transcarbamylase, while movement of the 80 loop and significant domain closure take place as in other transcarbamylases. These findings provide new information on the putative role of succinylated intermediates in arginine biosynthesis and on the evolution of transcarbamylases.     

HaloTag: a new versatile reporter gene system in plant cells

HaloTag Interchangeable Labeling Technology (HaloTag) was originally developed for mammalian cell analysis. In this report, the use of HaloTag is demonstrated in plant cells for the first time. This system allows different fluorescent colours to be used to visualize the localization of the non-fluorescent HaloTag protein within living cells. A vector was constructed which expresses the HaloTag protein under the control of the 35S promoter of cauliflower mosaic virus. The functionality of the HaloTag construct was tested in transient assays by (i) transforming tobacco protoplasts and (ii) using biolistic transformation of intact leaf cells of tobacco and poplar plants. Two to fourteen days after transformation, the plant material was incubated with ligands specific for labelling the HaloTag protein, and fluorescence was visualized by confocal laser scanning microscopy. The results demonstrate that HaloTag technology is a flexible system which generates efficient fluorescence in different types of plant cells. The ligand-specific labelling of HaloTag protein was not hampered by the plant cell wall.     

A common mode of attraction of larvae and adults of insect predators to the sex pheromone of their prey (Hemiptera: Matsucoccidae)

The attraction of several adult predators, genera Elatophilus, Hemerobius and Sympherobius, to the sex pheromones of pine bast scales, Matsucoccus Cockerell, has already been demonstrated. Here, the hypothesis that the larvae of these predators are similarly attracted to the host prey sex pheromone is tested. The response of predators was tested in field trials using pine tree arenas baited with the sex pheromones of M. josephi Bodenheimer & Harpaz, M. feytaudi Ducasse and M. matsumurae Kuwana. Experiments were conducted in Israel in stands of Pinus halepensis infested by M. josephi and in Portugal in stands of P. pinaster infested by M. feytaudi, respectively. The selectivity of larvae for the three sex pheromones was tested in Petri dish arenas in the laboratory. In the field, the larval stages exhibited similar modes of attraction to those of the conspecific adults: Elatophilus hebraicus Pericart in Aleppo pine forest, E. crassicornis Reuter and Hemerobius stigma Stephens in the maritime pine forests. Laboratory choice tests confirmed the kairomonal selectivity of larvae. Both forest and laboratory tests demonstrated the response of a coccinellid of the genus Rhyzobius to the sex pheromones of M. feytaudi and M. matsumurae. A unique chemical communication system among several taxa of predators of Matsucoccus spp. was highlighted that may be attributed to their coevolution on a geological time scale.     

Cell biology of molybdenum

The transition element molybdenum (Mo) is of essential importance for (nearly) all biological systems as it is required by enzymes catalyzing diverse key reactions in the global carbon, sulfur and nitrogen metabolism. The metal itself is biologically inactive unless it is complexed by a special cofactor. With the exception of bacterial nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor (Moco) which is the active compound at the catalytic site of all other Mo-enzymes. In eukaryotes, the most prominent Mo-enzymes are (1) sulfite oxidase, which catalyzes the final step in the degradation of sulfur-containing amino acids and is involved in detoxifying excess sulfite, (2) xanthine dehydrogenase, which is involved in purine catabolism and reactive oxygen production, (3) aldehyde oxidase, which oxidizes a variety of aldehydes and is essential for the biosynthesis of the phytohormone abscisic acid, and in autotrophic organisms also (4) nitrate reductase, which catalyzes the key step in inorganic nitrogen assimilation. All Mo-enzymes, except plant sulfite oxidase, need at least one more redox active center, many of them involving iron in electron transfer. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also includes iron as well as copper in an indispensable way. Moco as released after synthesis is likely to be distributed to the apoproteins of Mo-enzymes by putative Moco-carrier proteins. Xanthine dehydrogenase and aldehyde oxidase, but not sulfite oxidase and nitrate reductase, require the post-translational sulfuration of their Mo-site for becoming active. This final maturation step is catalyzed by a Moco-sulfurase enzyme, which mobilizes sulfur from l-cysteine in a pyridoxal phosphate-dependent manner as typical for cysteine desulfurases.     

The first record of a natural hybrid of the roach Rutilus rutilus and nase Chondrostoma nasus in the Danube River Basin, Czech Republic: morphological, karyological and molecular characteristics

Morphological (meristic and morphometric traits), karyological and molecular (microsatellites, cytochrome b ) analyses were performed to characterize a hybrid of the roach Rutilus rutilus and nase Chondrostoma nasus . Meristic and morphometric traits were different between hybrid and both parental species. The number of chromosomes found in hybrid specimen indicated that this individual represents the post-F₁ generation of hybrids and the microsatellite analysis of the hybrid showed the presence of variants typical for R. rutilus and C. nasus .     

Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind Moco with K_D values in the low micromolar range and are therefore named Moco-binding proteins (MoBP). For two of the nine proteins atomic structures are available in the Protein Data Bank. Surprisingly, both crystal structures lack electron density for the C terminus, which may indicate a high flexibility of this part of the protein. C-terminal truncated MoBPs showed significantly decreased Moco binding stoichiometries. Experiments where the MoBP C termini were exchanged among MoBPs converted a weak Moco-binding MoBP into a strong binding MoBP, thus indicating that the MoBP C terminus, which is encoded by a separate exon, is involved in Moco binding. MoBPs were able to enhance Moco transfer to apo-nitrate reductase in the Moco-free Neurospora crassa mutant nit-1. Furthermore, we show that the MoBPs are localized in the cytosol and undergo protein-protein contact with both the Moco donor protein Cnx1 and the Moco acceptor protein nitrate reductase under in vivo conditions, thus indicating for the MoBPs a function in Arabidopsis cellular Moco distribution.