Distribution of microtubules during the growth of tobacco pollen tubes

Summary— The distribution of microtubules was investigated in Nicotiana tabacum pollen tubes at different stages of tube growth by immunofluorescence microscopy. Using specific antibodies, the presence of microtubules consisting of different tubulin isoforms was tested. α-, β- and tyrosinated α-tubulin were present within the tube, whereas the acetylated form was lacking. The presence of tubulin subunits in pollen tube extracts was also investigated by immunoblotting analyses. The use of a confocal laser scanning microscope integrated with computer-assisted imaging, allowed a detailed visualization of the microtubule distribution and organization. Cytoplasmic microtubules organized as short bundles with various orientations were detected at the apex of long tubes.     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

The central region of the synaptonemal complex inBlaps cribrosa studied by electron microscope tomography

The synaptonemal complex (SC) in the beetleBlaps cribrosa contains a highly organized central element (CE), two flanking lateral elements (LEs), and a number of regularly spaced transverse filaments (TFs) crossing the central region. The CE is built like a ladder with two longitudinal components running in parallel and a number of regularly spaced transverse CE components, briding the two longitudinal components. The CE is multi-layered with the ladders of the individual layers more or less in register. Essentially every TF originates in one of the LEs, crosses the CE through a transverse CE component and reaches the opposite LE; every transverse CE component in a given layer corresponds to one, and only one, TF. In a CE layer, short irregular pillars form the junctions between the transverse and longitudinal CE components. Adjacent pillars are connected to each other by fine fibrous bridges: the two pillars in the same transverse CE component are linked, and so are the pillars along each longitudinal component, and also more occasionally adjacent pillars in separate CE layers. It is proposed that a TF with the two associated short pillars represents the structural unit in the central region. The ordered structure of the CE is accomplished by linking adjacent pillars to each other into the well-defined three-dimensional organization of the CE.     

Detection of a Novel Sepiapterin Reductase mRNA: Assay of mRNA in Various Cells and Tissues of Various Species

Fragments of cDNA coding for rat, murine, and human sepiapterin reductase (SR) were amplified by PCR via primer positioning close to the reported 3′-end of the coding region in the rat enzyme. They were sequenced and used as probes for mRNA detection. Northern blot analysis detected two mRNA species for SR. Their sizes were 1.3 and 2.1 kb for rat, 1.3 and 2.3 kb for mouse, and 1.6 and 2.1 kb for human cell lines. Comparison of rat cell lines and rat tissues indicated that in tissues only the 1.3-kb species is present. Washing of the Northern blots under different stringency conditions indicated a more stable interaction of the 1.3-kb mRNA species with the cDNA probe as compared to the 2.3-kb species. The 1.3-kb species corresponds to the reported 28.2-kDa molecular mass of rat SR monomer. SR mRNA expression is absent in the human NK-like cell line YT and in the murine erythroleukemia subclone B8/3, which both lack SR activity. Moreover, the relative mRNA expression correlates with the enzymatic activities of different cell lines within the same species. This indicates that SR activity is regulated by its steady state mRNA levels.     

Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134

2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 1341. It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis, cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite. Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide. Those lactones harbonring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.     

A novel type of meso-diaminopimelic acid-based peptidoglycan and novel poly (erythritol phosphate) teichoic acids in cell walls of two coryneform isolates from the surface flora of French cooked cheeses

The primary structure of the peptidoglycan and the teichoic acids of two coryneform isolates from the surface flora of French cooked cheeses, CNRZ 925 and CNRZ 926, have been determined. In the peptidoglycan, meso-diaminopimelic acid was localized in position three of the peptide subunit. It contained an d-glutamyl-d-aspartyl interpeptide bridge, connecting meso-diaminopimelic acid and d-alanine residues of adjacent peptide subunits. The -carboxyl group of d-glutamic acid in position two of peptide subunits was substituted with glycine amide. The teichoic acid pattern and composition differed between the strains: both contained an erythritol teichoic acid and strain CNRZ 925 also contained an N-acetylglucosaminylphosphate polymer. The erythritol teichoic acids differed in terms of the quality and quantity of substituents, but they both had N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid in common.Abbreviations DNP dinitrophenyl - Ery erythritol - Gal galactose - GlcN glucosamine - GlcNAc N-acetylglucosamine - GlcUANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid - Hex UANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyhexuronic - acid m-Dpm, meso-diaminopimelic acid - Mur muramic acid - MurNAc N-acetylmuramic acid     

Conversion of 2-chloromaleylacetate in Alcaligenes eutrophus JMP134

2,4-Dichlorophenoxyacetate (2,4-D) in Alcaligenes eutrophus JMP134 (pJP4) is degraded via 2-chloromaleylacetate as an intermediate. The latter compound was found to be reduced by NADH in a maleylacetate reductase catalyzed reaction. Maleylacetate and chloride were formed as products of 2-chloromaleylacetate reduction, the former being funnelled into the 3-oxoadipate pathway by a second reductive step. There was no indication for an involvement of a pJP4-encoded enzyme in either the reduction or the dechlorination reaction.Abbreviations 2,4-D 2,4-dichlorophenoxyacetate     

Tree seedling development in tropical dry abandoned pasture and secondary forest in Costa Rica

Development of planted seedlings of four canopy tree species in recently abandoned pastures (mown and unmown) and in ca. 40-yr old secondary dry forest in Guana-caste National Park, Costa Rica, was studied from July 1989–June 1992. The species were the light-demanding Cedrela odorata and Swietenia macrophylla, and the shade-tolerant Hymenaea courbaril and Manilkara chicle. Seedling mortality was high and primarily correlated with dry season drought. After the first dry season, M. chicle and C. odorata showed < 5 % survival in the pasture and 20 % in the forest, but after 3 yr survival had decreased to 3% in all plots. Survival of S. macrophylla was highest in the mown pasture (45 % after 3 yr) and lowest in the forest (10 %). For H. courbaril, survival was 40–55 % under all three conditions. Growth rates were low, with a height increment of < 15 cm/yr, possibly due to low precipitation in 1990 and 1991. All species grew taller in the pasture than in the forest. Differences in soil depth, texture and drainage appeared to contribute to variation in the results both within and between plots. Herbivory contributed to the lower survival of C. odorata and S. macrophylla in the forest.