Pyrimidine degradation in tomato cell suspension cultures and in Euglena gracilis—localization of enzymes

• 1.1. Subcellular location of dihydropyrimidinase and NCβA-amidohydrolase2 was studied in a cell suspension culture of tomato (Lycopersicon esculentum cv. Lukullus) and in Euglena gracilis.
• 2.2. By differential centrifugation, crude extracts were separated into ten fractions. Activities of both enzymes were found mainly in cytosolic fractions marked by EDH (tomato) and glu-6-P-DH (E. gracilis).
• 3.3. A cytosolic location was also found by a 20–60% and a 17.5–30% sucrose density gradients.
• 4.4. Using mitochondrial marker enzymes such as fumarase, SDH, CS and MDH, a mitochondrial occurrence of both enzymes or their release from mitochondria can be excluded by sucrose gradient centrifugations. This can also be achieved using purified mitochondria prepared from tomato cells by two subsequent sucrose gradients.
• 5.5. A possible vacuolar location of dihydropyrimidinase and NCβA-amidohydrolase was excluded by comparing their activities in isolated protoplasts and purified vacuoles which were characterized by their marker enzyme α-mannosidase.
• 6.6. A nuclear location of both enzymes and/or their release from the nucleus during procedures used cannot be excluded.
• 7.7. The results are discussed in relation to subcellular location to other pyrimidine-metabolizing enzymes in plant cells.

     

Growth and infectivity of callus cultures of tomato plants infected with a mycoplasma disease — Potato witches' broom

Callus tissues were derived from the stem of healthy tomato plants (Lycopersicum esculentum Mill. ev. Pr?honické) and of plants infected with potato witches' broom—a disease caused by mycoplasma. Callus cultures were established on modified fully synthetic media described byMorel (1948) and byMurashige andSkoog (1962). Callus cultures obtained from diseased plants were grown and subcultured on both media, growth in primary isolates from healthy plants took place on the Murashige and Skoog medium only. Growth of callus tissue derived from diseased plants was more vigorous even after several subcultivations in comparison with callus tissues isolated from healthy plants. Variations in the morphology in these callus cultures were not noted. Callus cells of diseased plants varied in size; they were about 50% larger than those from healthy ones. Implantation of primary and subcultivated callus tissues into tomato stems of healthy plants did not show any symptoms of infection on test plants.     

Imaging the boundaries—innovative tools for microscopy of living cells and real-time imaging

Recently, light microscopy moved back into the spotlight, which is mainly due to the development of revolutionary technologies for imaging real-time events in living cells. It is truly fascinating to see enzymes “at work” and optically acquired images certainly help us to understand biological processes better than any abstract measurements. This review aims to point out elegant examples of recent cell-biological imaging applications that have been developed with a chemical approach. The discussed technologies include nanoscale fluorescence microscopy, imaging of model membranes, automated high-throughput microscopy control and analysis, and fluorescent probes with a special focus on visualizing enzyme activity, free radicals, and protein–protein interaction designed for use in living cells.     

Reactive oxygen and nitrogen (ROS and RNS) species generation and cell death in tomato suspension cultures—<Emphasis Type="Italic">Botrytis cinerea</Emphasis> interaction

This article reports events connected to cell survival and Botrytis cinerea infection development in cell suspension cultures of two tomato cultivars which show different levels of susceptibility to the pathogen: cv. Corindo (more susceptible) and cv. Perkoz (less susceptible). In parallel changes in reactive oxygen (ROS) and nitrogen (RNS) species generation and in S-nitrosoglutathione reductase (GSNOR) activity were studied. In vivo staining methods with acridine orange (AO) and ethidium bromide (EB) as well as fluorescent microscopy were used to assess tomato and B. cinerea cells death. The biochemical studies of ROS and RNS concentrations in plant cell extract were complemented by in vivo ROS and nitric oxide (NO) imaging using nitro blue tetrazolium (NBT), diaminobenzidine (DAB) and diaminofluorescein diacetate (DAF-DA) staining methods, and confocal microscope technique. B. cinerea infection proceeded slower in Perkoz cell cultures. It was evidenced by measuring the pathogen conidia germination and germination tube development in which nuclei revealing cell death dominated. Two different types of tomato cell death were observed: cells with necrotic nuclei dominated in Corindo whereas in Perkoz cells with characteristic of vacuolar death type prevailed. In Perkoz cells, constitutive levels of NO and S-nitrosothiols (SNO) were significantly higher and hydrogen peroxide (H₂O₂) and superoxide anion (O₂ ^?) concentrations were slightly higher as compared with Corindo cells. Moreover, increases in these molecule concentrations as a result of B. cinerea inoculation were observed in both, Perkoz and Corindo cell cultures. The enzymatic GSNOR activity seems to be an important player in controlling the SNO level in tomato cells. Involvements of the studied compounds in molecular mechanisms of tomato resistance to B. cinerea are discussed in the paper.     

Cluster analysis as a method for evaluation of genetic similarity in specific host — parasite interaction (Lactuca sativa — Bremia lactucae)

Summary In gene-for-gene systems, specificity of hostparasite interactions is most often estimated qualitatively using the symbols +, –, (i.e. susceptibility and/or resistance). In large sets (interaction patterns) it becomes impossible to analyze numerous data by mere comparison. This is overcome by application of cluster analysis. In our experiments the methods in question were used to estimate the data obtained in a study on interactions between more than 220 Lactuca sativa cultivars and 12 Bremia lactucae physiological races (isolates) of Czechoslovak origin. The matrix of similarity coefficients was analyzed by hierarchical clustering. Similarity and/or dissimilarity of host R-genotypes was graphically expressed using the method of two principal components. The results obtained are related to genetic constitution of race specific resistance of the host and the possibility of predicting effective resistance sources.     

Multinucleated giant cells in primary cultures derived from canine bone marrow—evidence for formation of putative osteoclasts

Summary Mononucleated cells derived from canine bone marrow were maintained in vitro for up to 6 weeks. The culture characteristics and development of these cells were evaluated by histological, ultrastructural and histochemical methods. Within 1 week the cells had fused together to form flattened, multinucleated cells. Further fusing with one another and other mononucleated cells produced large (diameters more than 300 m), multinucleated cells which frequently contained more than 50 nuclei per cell and exhibited ultrastructural and histochemical features that were strikingly similar to those displayed by osteoclasts. The confluent monolayer of mono- and multinucleated cells present at 4 weeks had, by the sixth week, become altered such that fibroblast overgrowth obliterated all other cells. During the development of the culture adipocytes became differentiated from mononuclear cells and frequently were located within spherical multicellular aggregates (spheroids). Functional assessments were employed to investigate whether the multinucleated cells generated in this way, represented osteoclast-like cells, or alternatively, were related to macrophage polykarya as found in foreign body granulomata in vivo. Neither resorption pits on sperm whale dentine slivers (diagnostic of osteoclasts), nor formation of granulomata in vitro, were observed. We believe that the present results indicate that the multinucleated cells generated from canine bone marrow mononuclear precursors in vitro, merit designation as osteoclast-like cells. Definitive characterisation however, must await further functional assessments of hormone responsiveness.Dr. Martin Bird died during the final phase of this work and it is to his memory that this paper is respectfully dedicated