GROWTH STUDIES OF THREE CHLOROPHYLLOUS CALLUS PHENOTYPES OF GLYCINE MAX

The establishment of three chlorophyllous callus phenotypes, Glycine max (L.) Merrill, cultured on a modified Miller's medium is described. Experiments were designed to determine the hormonal requirement necessary to maintain an adequate callus growth rate that would allow for the phenotypical accumulation of chlorophyll in all phenotypes. Addition of α-naphthaleneacetic acid and kinetin, both at 1 mg/liter, to the basal medium fulfilled this requirement. However, callus growth for these phenotypes required only an exogenous supply of cytokinin. All callus phenotypes, when maintained on 3% sucrose, were shown to possess similar growth curves; however, optimal growth rates of these cultured phenotypes occurred on different levels of exogenous sucrose (NG, 2%; LG, 1%; Y, 2%). Sucrose (filtered and autoclaved) and, in most cases, fructose (filtered), when employed as a carbon source in the basal medium, maintained adequate growth rates. Glucose (filtered) supported only minimal callus growth. These callus phenotypes, after two years in culture, showed a certain degree of cell type differentiation as indicated by the formation of isolated tracheoids and in some cases organized tracheoid development. The chromosome complement (2n = 40) was observed to be polyploid.     

The cytoskeletal architecture of Trypanosoma brucei

The cytoskeleton of Trypanosoma brucei has been analyzed by the high-resolution technique of quick-freeze deep-etch rotary-shadowing electron microscopy. The study provides detailed structural information on the subpellicular array of microtubules, the flagellum, and the interaction of these 2 major structures of the trypanosomal cytoskeleton with each other. The subpellicular microtubules closely interact both with the cell membrane and with each other. At the anterior tip of the cell they converge into a tightly closed structure, whereas at the posterior end the microtubular array remains open ended. The microtubular array is involved also in forming the opening of the flagellar pocket. The microtubular array interacts with the paraflagellar rod of the flagellum through a dense meshwork of fibers that are anchored on the microtubular surface with one end and within the paraflagellar rod structure with the other. The highly ordered, 3-dimensional network of the paraflagellar rod itself is connected tightly to the microtubular axoneme of the flagellum through a regular array of fleur-de-lis-shaped linking structures.     

Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions

This paper describes an adhesion/invasion assay, based on combined pyrrolidine dithiocarbamate (PDTC) and antibody treatment of parasites followed by quantitative real-time PCR. This PDTC-PCR assay can be used to comparatively assess the participation of host cell- and parasite-associated components during host cell adhesion and entry by Neospora caninum and Toxoplasma gondii tachyzoites, respectively, and is potentially applicable to any other apicomplexan parasite. The assay allows to determine the parasite invasion rate in relation to the overall number of parasites which interact with host cells in any given experiment, and thus represents a significant improvement to conventional microscopic assays in terms of accuracy and reproducibility. Using this assay it was possible to show that adhesion and invasion of N. caninum tachyzoites are two distinct and separated events, in that N. caninum tachyzoites preferentially utilise host cell surface chondroitin sulphates for adhesion, but not for the host cell invasion process. Application of the PDTC-PCR assay also demonstrated that N. caninum and T. gondii tachyzoites differ largely with regard to the functional involvement of proteases in adhesion and invasion of host cells. Thus, although phylogenetically closely related, N. caninum and T. gondii are biologically quite different and exhibit distinct dissimilarities with regard to host cell interactions.     

Reduced cerebral infection of Neospora caninum-infected mice after vaccination with recombinant microneme protein NcMIC3 and ribi adjuvant

C57BL/6 mice were vaccinated with a bacterially expressed and purified polyhistidine-tagged full-length version of the microneme protein NcMIC3 (recNcMIC3) emulsified in Ribi Adjuvant System (RAS). Subsequently, they were challenged by intraperitoneal inoculation of 2 x 10(6) live Neospora caninum tachyzoites. As controls, groups of mice received phosphate-buffered saline (PBS)-RAS alone (adjuvant control) or were treated with PBS before infection (infection control). The protective effect of vaccination was assessed by Neospora-specific polymerase chain reaction (PCR), immunohistochemical investigation of brain tissue, and serological means (enzyme-linked immunosorbent assay). Assessment by PCR performed on DNA from different organs revealed that in all treatment groups parasite DNA could only be detected in brain tissue. According to the PCR results. the recNcMIC3 vaccine conferred protection to 75% of mice (n = 16 in 2 independent experiments), whereas application of PBS-RAS and of PBS alone resulted in protection of 12.5% and 0% of mice, respectively (n = 16 as above). Mice in the PBS-treated infection control group were affected by evident clinical signs of neosporosis starting on day 6 postinfection (p.i.). Conversely, none of the animals treated with either PBS-RAS or recNcMIC3 exhibited any symptoms until day 21 p.i. Immunohistochemical staining of paraffin-embedded brain tissue sections confirmed the protective effect of recNcMIC3 vaccination. Quantitative Neospora-specific real-time PCR revealed that infection intensities were lower in the brain tissues of recNcMIC3-vaccinated mice compared with PBS-RAS-treated adjuvant control mice. Serological analysis showed that the protective effect observed in recNcMIC3-vaccinated mice was associated with a Th2-type IgG1 antibody response directed against native NcMIC3 and a mixed IgG1-IgG2a antibody response directed against the recombinant antigen itself. Taken together, these results demonstrated that recombinant NcMIC3 vaccine confers a significant protectivity against experimentally induced cerebral neosporosis in mice.     

Competition between two stream dwelling filter-feeders,Hydropsyche oslari and Simulium virgatum

Summary This study investigated whether interspecific competition affected the abundance and distribution of Hydropsyche oslari and Simulium virgatum. In addition, I studied the mechanisms of competition between the two taxa. H. oslari was the superior competitor when compared to S. virgatum because the presence of H. oslari in boulder habitats in Refugio Creek altered the microdistribution and depressed numbers of S. virgatum. Hydropsyche preempted space and was aggressive towards Simulium. Simulium avoided both hydropsychid larvae and their nets. This avoidance behaviour was reinforced by aggression from H. oslari. Possible immediate reasons why simuliids avoided Hydropsyche nets included 1) Hydropsyche attacked Simulium, 2) occupied nets interfered with feeding through increased turbulence or lower renewal rate for food, and 3) Simulium preferred to settle with conspecifics.