Detection of Lewis(a) antigen in sera of ovarian carcinoma patients by MOv2-MOv8 double-determinant radioimmunoassay

The monoclonal antibodies MOv2 and MOv8, raised against ovarian carcinoma, were found to be directed against two non-crossreacting epitopes expressed on the same molecule. Immunochemical analysis of the MOv8 recognized epitope showed that the Le(a) oligosaccharide, or commercial anti-Le(a) MAb, but not the anti-Le(b) MAb, prevented Ov8 binding to the reference target cell line (SW626), indicating that it is carried by the Le(a) antigen. Since we previously reported that MOv2 also recognises the Le(a) antigen, these data suggest that Mov8 and Mov2 were directed against different epitopes on the same oligosaccharide chain. Bearing in mind the knowledge of the biochemical nature of the monoclonal antibody recognized epitopes (CaMOv2 and CaMOv8), the presence of the circulating molecules recognized by them was analyzed by double determinant immunoradiometric assay (DDIRMA) in 103 sera from ovarian carcinoma patients. Patients with clinical evidence of the disease (ED) with MOv2 and MOv8 reactive and negative tumors had sera reactivity in 67% and 19% respectively. Also, 26% of the patients with no clinical evidence of disease (NED) had positive sera. When we investigated the relationship between MOv2-MOv8 DDIRMA sera positivity and red blood cells (RBC) Lewis phenotype, a strong correlation was found between the Le(a)+ phenotype and DDIRMA sera reactivity in healthy donors (6/6) and in ovarian carcinoma patients (9/10) whatever their clinical condition. No Le(a)- healthy donors gave evidence of MOv2-MOv8 reactive sera. In contrast, 33% and 57% of the sera from ED carcinoma patients with respectively Lea-b+ and Lea-b- phenotype were positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a Real-time Polymerase Chain Reaction for the Detection of Fusarium oxysporum f. sp. basilici from Basil Seed and Roots

A real‐time polymerase chain reaction (PCR) assay using a TaqMan^® probe was developed to detect the causal agent of wilt and crown rot of basil from infected plants and seed in Italy. The aim of the study was to diminish testing time, previously performed using nested‐PCR, and to create the conditions for future automation. The sensitivity of the assay was shown to be similar to the detection limit of the available nested‐PCR procedure. The advantages of real‐time PCR system include halving of the testing time, as well as the ability to identify both internally and externally infected seed to the sensitivity of 1 pg of genomic DNA. The assay was able to detect the presence of the pathogen in infected seed up to a sensitivity of 24 (SD: ±10) CFU per 100 seeds.     

Subcellular fractionation, electromigration analysis and mapping of organelles

Subcellular fractionation has provided the means required to analyze the composition and properties of purified cellular elements. In particular, subcellular fractionation has helped to define membrane boundaries and became necessary for the development of cell-free assays that reconstitute complicated cellular processes. Although cell fractionation techniques have improved over the last decades the purification of organelles to homogeneity is still a barely accessible goal in cell biology. In this article, we will first briefly review the basic principles of subcellular fractionation, and the establishment of different organelle fractions by density centrifugation, using tissue culture cells as a paradigm. Then we will discuss some of the intrinsic problems and will compare gradient purification of cellular extracts with electromigration analysis. Finally, we will describe alternative approaches, such as immunoisolation and flow cytometry to purify organelles from tissue culture cells.     

Differential in vitro phenotype pattern, transforming growth factor-β1 activity and mRNA expression of transforming growth factor-β1 in Apert osteoblasts

The phenotype of Apert osteoblasts differs from that of normal osteoblasts in the accumulation of macromolecules in the extracellular matrix. Apert osteoblasts increase type I collagen, fibronectin and glycosaminoglycans secretion compared with normal osteoblasts. Because the extracellular matrix macromolecule accumulation is greatly modulated by transforming growth factor-beta(1), we examined the ability of normal and Apert osteoblasts to secrete transforming growth factor-beta(1) by CCL-64 assay and to produce transforming growth factor-beta(1 )by analysis of the mRNA expression of transforming growth factor-beta(1). Northern blot analysis revealed an increased amount of transforming growth factor-beta(1) mRNA expression in Apert osteoblasts compared with normal ones. Moreover, the level of the active transforming growth factor-beta(1) isoform was higher in Apert than in normal media. In pathologic cells, the increase in transforming growth factor-beta(1) gene expression was associated with a parallel increase in the factor secreted into the medium. The level of transforming growth factor-beta(1) was decreased by the addition of basic fibroblast growth factor. Transforming growth factor-beta(1) is controlled temporally and spatially during skeletal tissue development and produces complex stimulatory and inhibitory changes in osteoblast functions. We hypothesise that in vitro differences between normal and Apert osteoblasts may be correlated to different transforming growth factor-beta(1) cascade patterns, probably due to an altered balance between transforming growth factor-beta(1) and basic fibroblast growth factor.     

Cell-cycle kinetics of friend erythroleukemia cells in a magnetically shielded room and in a low-frequency/low-intensity magnetic field

This work was undertaken to compare the behavior of Friend erythroleukemia cells in a solenoid, where the magnetic field was 70 μT at 50 Hz (plus 45 μT DC of Earth) with that of the same cells in a magnetically shielded room, where the magnetic field was attenuated to 20 nT DC and 2.5 pT AC. The control laboratory magnetic field corresponded to 45 μT DC and a stray 50 Hz field below 0.2 μT. The culture growth cycle of cells maintained inside the solenoid was slightly accelerated compared with that of cells maintained outside the solenoid (P < .05). This stimulation probably depended on sensitivity of cell cycle to a magnetic field, because, inside the solenoid, the percentage of G₁ cells slightly increased during the culture growth cycle, whereas that of S cells slightly decreased. Acceleration of growth was detected soon after exposure of the cultures to the solenoid field, and growth did not change further if the action of this field continued for a long time, accounting for adaptation. The solenoid field also caused a small increase of cell survival without influencing cell volume. By contrast, the culture growth cycle of cells maintained inside the magnetically shielded room was slightly decelerated compared with that of cells maintained outside the room (P < .05). The essential absence of any field inside the magnetically shielded room also caused a small increase of cell volume, whereas, during the culture growth cycle, the percentage of G₁ cells decreased, and that of S cells increased. The majority of these events did not change in cells induced to differentiate hemoglobin through dimethylsulfoxide. Bioelectromagnetics 18:58–66, 1997. © 1997 Wiley-Liss, Inc.     

Calcium opens the dialogue between plants and arbuscular mycorrhizal fungi

Calcium ion is considered a ubiquitous second messenger in all eukaryotic cells. Analysis of intracellular Ca²⁺ concentration dynamics has demonstrated its signalling role in plant cells in response to a wide array of environmental cues. The implication of Ca²⁺ in the early steps of the arbuscular mycorrhizal symbiosis has been frequently claimed, mainly by analogy with what firmly demonstrated in the rhizobium-legume symbiosis. We recently documented transient Ca²⁺ changes in plant cells challenged with diffusible molecules released by arbuscular mycorrhizal fungi. Ca²⁺ measurements by the recombinant aequorin method provided new insights into the molecular communications between plants and these beneficial fungi.Key words: legume symbioses, arbuscular mycorrhiza, calcium signalling, fungal signal, plant cell cultures, aequorinIn the rhizosphere plants meet a wide array of microorganisms. In favorable interactions, such as arbuscular mycorrhizal (AM) and nitrogen fixing symbioses, a dialogue is progressively established between the two interacting organisms to make the appropriate partner choice. These two-way communications rely on the interchange of signals released by both potential symbionts. After perception of the signalling molecules, a signal transduction pathway is induced, leading to the activation of the proper genetic and developmental program in both partners.Variations in intracellular free Ca²⁺ concentration occur as one of the initial steps in signalling pathways activated in plants when they encounter pathogens,¹ fungal biocontrol agents² and nitrogen-fixing bacteria.³ Molecules secreted by microorganisms, after binding to specific receptors, trigger in plant cells transient changes in cytosolic Ca²⁺ level, due to the influx of the ion from the extracellular environment and/or the release from internal Ca²⁺ storage compartments.^4,5 Ca²⁺ messages delivered to plant cells are at least partly deciphered on the basis of their spatial and temporal features. The occurrence of different Ca²⁺ signatures guarantees the specificity of the ensuing physiological responses.In the legume-rhizobium symbiosis a definite pattern of Ca²⁺ oscillations has been reported to occur in response to the rhizobial signalling molecule, the Nod factor, in the nucleus and perinuclear cytoplasm of the root hair.⁶ The Ca²⁺ spike number has been recently demonstrated to regulate nodulation gene expression.⁷Legumes are able to engage in a dual symbiotic interaction, with rhizobia and AM fungi. Components of the Ca²⁺-mediated signalling pathway are shared by the two symbioses.⁸ In the mycorrhizal signal transduction pathway the involvement of Ca²⁺ has long been speculated, based on the observed similarities with symbiotic nitrogen fixation.³To evaluate the possible participation of Ca²⁺ in the early steps of the AM symbiosis, we have used a simplified experimental system given by plant cell suspension cultures stably expressing the bioluminescent Ca²⁺-sensitive reporter aequorin.⁹ The use of cultured cells circumvents the problem posed by multilayered organs: in aequorin-transformed seedlings, possible Ca²⁺ changes occurring in rhizodermal cells—the first place where the AM fungal signals are perceived and transduced—can be misrecorded due to luminescence calibration over all root cell layers, resulting in an underestimation of the Ca²⁺ signal in the responsive cells. An experimental design based on challenging host plant cells with the culture medium of different AM fungi (Gigaspora margarita, Glomus mosseae and intraradices) provided the first firm evidence that Ca²⁺ is involved as intracellular messenger during mycorrhizal signalling, at least in a pre-contact stage. Cytosolic Ca²⁺ changes, characterized by specific kinetic parameters, were triggered by diffusates obtained from AM resting and germinating spores,⁹ and extraradical mycelium.¹⁰ Cultured plant cells demonstrated to be competent to perceive the diffusible signal released by AM fungi and to decode the message in a Ca²⁺-dependent pathway. Based on these experiments, it seems that AM fungi announce their presence to the plant through the constitutive release of a chemical signal, even before experiencing the proximity of the plant or its AM symbiotic signals. The notion that the secreted fungal molecules herald, through Ca²⁺, a beneficial message which can be acknowledged only by competent receivers, is supported by: (1) the lack of defense response induction and the upregulation of some genes essential for the AM symbiosis initiation in host plant cells; (2) the unresponsiveness of cultured cells from the nonhost plant Arabidopsis thaliana.Ca²⁺-mediated perception of both AM fungal and rhizobial signals by plant cells unifies the signalling pathways activated in the two symbioses. However, the actual occurrence of Ca²⁺ spiking in AM symbiosis remains to be ascertained, due to limitations of the recombinant aequorin method, when applied to an asynchronous cell population. Contribution of internal Ca²⁺ stores, in particular the nucleus, to the observed Ca²⁺ changes will be a future research goal to be achieved through a pharmacological approach and/or targeting of Ca²⁺ indicators to intracellular compartments.The identification of the plant-derived mycorrhizal signal as strigolactones¹¹ and their inducing activity on AM fungi¹² have represented a major breakthrough in the AM symbiosis research field. Elucidation of the chemical nature of the AM fungal factor, which plays several effects on host plants,^9,13–15 is eagerly awaited.Understanding how AM fungi and rhizobia select compatible plant hosts, thus activating the appropriate symbiotic program, is another facet to be considered in the future to get a complete overview of early signaling events in legume symbioses. Analysis of Ca²⁺ signalling implication in the microbial partner would require the delivery of reliable and sensitive Ca²⁺ probes (such as aequorinor GFP-based¹⁶) for Ca²⁺ measurements in living microorganisms. The recombinant aequorin method has been successfully applied to monitor dynamic changes in intracellular Ca²⁺ levels in the bacteria Anabaena sp.,¹⁷ E. coli,¹⁸ and recently by us in rhizobial strains.¹⁹ Unfortunately, AM fungi have proved not to be amenable to stable transformation, being coenocytic, multinucleate and heterokaryotic,^20,21 and only transient transformants have been obtained so far.^22,23 Further development of the transformation technologies may provide in the future a valuable tool to analyse, from the fungal side, signal perception and transduction during arbuscular mycorrhiza establishment.     

Increased Receptor for Advanced Glycation Endproducts Immunocontent in the Cerebral Cortex of Vitamin A-Treated Rats

Vitamin A, beyond its biological role, is an alternative choice in treating some life threatening pathologies, for instance leukemia and immunodeficiency. On the other hand, vitamin A therapy at moderate to high doses has caused concern among public health researchers due to the toxicological aspect resulting from such habit. It has been described hepatotoxicity, cognitive disturbances and increased mortality rates among subjects ingesting increased levels of vitamin A daily. Then, based on the previously reported data, we investigated here receptor for advanced glycation endproducts (RAGE) immunocontent and oxidative damage levels in cerebral cortex of vitamin A-treated rats at clinical doses (1,000–9,000 IU/kg day⁻¹). RAGE immunocontent, as well as oxidative damage levels, were observed increased in cerebral cortex of vitamin A-treated rats. Whether increased RAGE levels exert negative effects during vitamin A supplementation it remains to be investigated, but it is very likely that deleterious consequences may arise from such alteration.