Secretion systems and membrane-associated structures in rust fungi after high pressure freezing and freeze-fracturing

Excellent preservation and new structural details can be demonstrated in rust-infected leaf tissue after high pressure freezing and freeze-fracturing. A tubular-vesicular complex was the most remarkable cytoplasmic structure observed in cells of the bean rust fungus Uromyces appendiculatus var. appendiculatus during its establishement in its host Phaseolus vulgaris. In fungal cells undergoing intensive synthesis of wall material, this membranous system extended throughout the cytoplasm; in addition, vesicles were accumulated adjacent to the plasma membrane. Here, membrane-associated configurations were observed which seem to be involved in exo- and/or endocytotic processes. It is assumed that the tubular-vesicular complex belongs to the endomembraneous system of the bean rust fungus and that it is involved in the synthesis and secretion of wall material.     

3-([3-125I]iodo-4-hydroxyphenyl)propionyl carbohydrazide,a new radioiodination reagent for ultrasensitive detection and determination of periodate-oxidized nucleoside derivatives and other carbonyl compounds

A new radioiodination reagent for the identification and quantitation of periodate-oxidized ribonucleosides was developed. The reagent, 3-([3-¹²⁵I]iodo-4-hydroxyphenyl)propionyl carbohydrazide, was prepared by radioiodination of 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester in the presence of chloramine T, followed by reduction of the latter with sodium arsenite and treatment of the radioiodinated ester with an excess of carbohydrazide. The reagent reacted quantitatively with periodate-oxidized nucleosides to form ¹²⁵I-labeled morpholine derivatives which were separated by thin-layer chromatography and quantitated by liquid scintillation counting. The reagent was found to react also with other carbonyl compounds and thus may find more general application in the qualitative and quantitative ultramicroanalysis of aldehydes and ketones.     

Conformational analysis of protein and nucleic acid fragments with the new grid search algorithm FOUND

The new computer algorithm FOUND, which is implemented as an integrated module of the DYANA structure calculation program, is capable of performing systematic local conformation analyses by exhaustive grid searches for arbitrary contiguous fragments of proteins and nucleic acids. It uses torsion angles as the only degrees of freedom to identify all conformations that fulfill the steric and NMR-derived conformational restraints within a contiguous molecular fragment, as defined either by limits on the maximal restraint violations or by the fragment-based DYANA target function value. Sets of mutually dependent torsion angles, for example in ribose rings, are treated as a single degree of freedom. The results of the local conformation analysis include allowed torsion angle ranges and stereospecific assignments for diastereotopic substituents, which are then included in the input of a subsequent structure calculation. FOUND can be used for grid searches comprising up to 13 torsion angles, such as the backbone of a complete -helical turn or dinucleotide fragments in nucleic acids, and yields a significantly higher number of stereospecific assignments than the precursor grid search algorithm HABAS.     

Association between plasma antibody response and protection in rainbow trout Oncorhynchus mykiss immersion vaccinated against Yersinia ruckeri

A key hallmark of the vertebrate adaptive immune system is the generation of antigen-specific antibodies from B cells. Fish are the most primitive gnathostomes (jawed vertebrates) possessing an adaptive immune system. Vaccination of rainbow trout against enteric redmouth disease (ERM) by immersion in Yersinia ruckeri bacterin confers a high degree of protection to the fish. The immune mechanisms responsible for protection may comprise both cellular and humoral elements but the role of specific immunoglobulins in this system has been questioned and not previously described. The present study demonstrates significant increase in plasma antibody titers following immersion vaccination and significantly reduced mortality during Y. ruckeri challenge.Rainbow trout were immersion-vaccinated, using either a commercial ERM vaccine (AquaVac™ ERM vet) or an experimental Y. ruckeri bacterin. Half of the trout vaccinated with AquaVac™ ERM vet received an oral booster (AquaVac™ ERM Oral vet). Sub-groups of the fish from each group were subsequently exposed to 1x10⁹ CFU Y. ruckeri/ml either eight or twenty-six weeks post vaccination (wpv). All vaccinated groups showed 0% mortality when challenged, which was highly significant compared to the non-vaccinated controls (40 and 28% mortality eight and twenty-six weeks post vaccination (wpv), respectively) (P<0.0001). Plasma samples from all groups of vaccinated fish were taken 0, 4, 8, 12, 16 and 26 wpv. and Y. ruckeri specific IgM antibody levels were measured with ELISA. A significant increase in titers was recorded in vaccinated fish, which also showed a reduced bacteremia during challenge. In vitro plasma studies showed a significantly increased bactericidal effect of fresh plasma from vaccinated fish indicating that plasma proteins may play a role in protection of vaccinated rainbow trout.     

Fragmentation is crucial for the steady-state dynamics of actin filaments

Despite the recognition that actin filaments are important for numerous cellular processes, and decades of investigation, the dynamics of in vitro actin filaments are still not completely understood. Here, we follow the time evolution of the length distribution of labeled actin reporter filaments in an unlabeled F-actin solution via fluorescence microscopy. Whereas treadmilling and diffusive length fluctuations cannot account for the observed dynamics, our results suggest that at low salt conditions, spontaneous fragmentation is crucial.     

Endocytosis of 1,3-β-glucans by broad bean cells at the penetration site of the cowpea rust fungus (haploid stage)

Te penetration hypha of basidiospore-derived infection structures of the cowpea rust fungus (Uromyces vignae Barclay) in epidermal cells of the nonhost, broad bean (Vicia faba L.), was studied with the electron microscope after high-pressure freezing and freeze substitution. After fungal invasion of the epidermis, a plug in the penetration hypha separated the infection structures on the cuticle from the intraepidermal vesicle of the fungus. The plug and the fungal cell wall reacted with a polyclonal 1,3-β-glucan antibody. The plug in the haploid stage seems to have a task similar to the septum formed in the diploid stage of the fungus. Around the penetration hypha, the plant wall stained darkly and a papilla was deposited by the plant. In the papilla, 1,3-β-glucans were labelled by a monoclonal and a polyclonal antibody. In the infected epidermal cell, clathrin-coated pits, coated vesicles, partially coated reticula and multivesicular bodies were found. The contents of the coated pits, coated vesicles, partially coated reticula and multivesicular bodies bound to monoclonal and polyclonal 1,3-β-glucan antibodies. Accumulation and uptake of this paramural material into the plant cell by endocytosis is concentrated at the fungal penetration site. It may influence the host-parasite interaction.     

Synthesis of α-mannosylated ergot alkaloids employing α-mannosidase

The ergot alkaloids elymoclavine, ergometrine and chanoclavine were α-mannosylated with α-mannosidase as catalyst. The kinetic reaction with p-nitrophenyl α-mannoside as glycosyl donor gave ca 28 % yield of chanoclavine α-mannoside, whereas the equilibrium reaction with mannose as the glycosyl donor gave ca 11 % yield. However, in the case of elymoclavine and ergometrine, higher yields of α-mannosides were obtained with the equilibrium approach (18 and 13 %).