Immunocytological Approaches Research Articles

Visualization of Chromatin in the Yeast Nucleus and Nucleolus Using Hyperosmotic Shock.

Unlike in most eukaryotic cells, the genetic information of budding yeast in the exponential growth phase is only present in the form of decondensed chromatin, a configuration that does not allow its visualization in cell nuclei conventionally prepared for transmission electron microscopy. In this work, we studied the distribution of chromatin and its relationships to the nucleolus using different cytochemical and immunocytological approaches applied to yeast cells subjected to hyperosmotic shock. Our results show that osmotic shock induces the formation of heterochromatin patches in the nucleoplasm and intranucleolar regions of the yeast nucleus. In the nucleolus, we further revealed the presence of osmotic shock-resistant DNA in the fibrillar cords which, in places, take on a pinnate appearance reminiscent of ribosomal genes in active transcription as observed after molecular spreading (“Christmas trees”). We also identified chromatin-associated granules whose size, composition and behaviour after osmotic shock are reminiscent of that of mammalian perichromatin granules. Altogether, these data reveal that it is possible to visualize heterochromatin in yeast and suggest that the yeast nucleus displays a less-effective compartmentalized organization than that of mammals.

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning.

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 μM) and high (50 μM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

Ultrastructural detection of nucleic acids within heat shock-induced perichromatin granules of HeLa cells by cytochemical and immunocytological methods

The perichromatin granules (PGs) are enigmatic structures of the cell nucleus. The major drawbacks for a biological study are their rare occurrence and their small size in normal conditions. As heat shock has been shown to increase their number, we applied a hyperthermal shock on HeLa cells to investigate the nucleic acid content of PGs by means of cytochemical and immunocytological approaches. These heat shock-induced PGs (hsiPGs) appeared as clusters organized in the form of honeycomb structures and were always associated with some blocks of condensed chromatin, such as the perinucleolar chromatin shell. A stalk connecting the hsiPG to the chromatin could be observed. For the detection of RNA, we applied an immunocytological method involving two anti-RNA antibodies and quantified the gold labelling obtained. The results clearly revealed that hsiPGs contained RNA. Regarding to the detection of DNA, we used three different methods followed by quantitative analyses. The results seemed to indicate that a small amount of DNA was present in hsiPGs. Together, these findings suggest that hsiPGs might be RNP structures associated with particular regions of DNA.

The kinetoplast ultrastructural organization of endosymbiont-bearing trypanosomatids as revealed by deep-etching, cytochemical and immunocytochemical analysis

The endosymbiont-bearing trypanosomatids present a typical kDNA arrangement, which is not well characterized. In the majority of trypanosomatids, the kinetoplast forms a bar-like structure containing tightly packed kDNA fibers. On the contrary, in trypanosomatids that harbor an endosymbiotic bacterium, the kDNA fibers are disposed in a looser arrangement that fills the kinetoplast matrix. In order to shed light on the kinetoplast structural organization in these protozoa, we used cytochemical and immunocytological approaches. Our results showed that in endosymbiont-containing species, DNA and basic proteins are distributed not only in the kDNA network, but also in the kinetoflagellar zone (KFZ), which corresponds to the region between the kDNA and the inner mitochondrial membrane nearest the flagellum. The presence of DNA in the KFZ is in accordance with the actual model of kDNA replication, whereas the detection of basic proteins in this region may be related to the basic character of the intramitochondrial filaments found in this area, which are part of the complex that connects the kDNA to the basal body. The kinetoplast structural organization of Bodo sp. was also analyzed, since this protozoan lacks the highly ordered kDNA-packaging characteristic of trypanosomatid and represents an evolutionary ancestral of the Trypanosomatidae family.

Occurrence of the allene oxide cyclase in different organs and tissues of Arabidopsis thaliana

Occurrence of an essential enzyme in jasmonate (JA) biosynthesis, the allene oxide cyclase, (AOC) was analyzed in different developmental stages and various organs of Arabidopsis thaliana plants by immuno blot analysis and immunocytological approaches. Levels of AOC and of the two preceding enzymes in JA biosynthesis increased during seedling development accompanied by increased levels of JA and 12-oxophytodienoic acid levels after 4 and 8 weeks. Most tissues including all vascular bundles and that of flower buds contain AOC protein. Flowers shortly before opening, however, contain AOC protein preferentially in ovules, stigma cells and vascular bundles, whereas in anthers and pollen AOC could not be detected. The putative roles of AOC and JA in development are discussed.

Patterns of Meiotic Recombination in Human Fetal Oocytes

Abnormal patterns of meiotic recombination (i.e., crossing-over) are believed to increase the risk of chromosome nondisjunction in human oocytes. To date, information on recombination has been obtained using indirect, genetic methods. Here we use an immunocytological approach, based on detection of foci of a DNA mismatch-repair protein, MLH1, on synaptonemal complexes at prophase I of meiosis, to provide the first direct estimate of the frequency of meiotic recombination in human oocytes. At pachytene, the stage of maximum homologous chromosome pairing, we found a mean of 70.3 foci (i.e., crossovers) per oocyte, with considerable intercell variability (range 48-102 foci). This mean equates to a genetic-map length of 3,515 cM. The numbers and positions of foci were determined for chromosomes 21, 18, 13, and X. These chromosomes yielded means of 1.23 foci (61.5 cM), 2.36 foci (118 cM), 2.5 foci (125 cM), and 3.22 foci (161 cM), respectively. The foci were almost invariably located interstitially and were only occasionally located close to chromosome ends. These data confirm the large difference, in recombination frequency, between human oocytes and spermatocytes and demonstrate a clear intersex variation in distribution of crossovers. In a few cells, chromosomes 21 and 18 did not have any foci (i.e., were presumptively noncrossover); however, configurations that lacked foci were not observed for chromosomes 13 and X. For the latter two chromosome pairs, the only instances of absence of foci were observed in abnormal cells that showed chromosome-pairing errors affecting these chromosomes. We speculate that these abnormal fetal oocytes may be the source of the nonrecombinant chromosomes 13 and X suggested, by genetic studies, to be associated with maternally derived chromosome nondisjunction.

Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes.

Two types of porcine circovirus (PCV) have been isolated and are referred to as PCV1 and PCV2. PCV1 represents an apathogenic virus, whereas PCV2 is associated with post-weaning multisystemic wasting syndrome. The two PCVs are related, since they display about 70% identity based on nucleotide sequences. In order to discriminate between common and type-specific antigens, an immunocytological approach was used following transfections with cloned circovirus DNAs, as well as recombinant proteins expressed by either baculovirus or plasmid vectors. The ORF1-encoded proteins in the two viruses were shown to be antigenically related, whereas the ORF2 proteins were recognized differentially by polyclonal anti-PCV2 antibodies. Furthermore, PEPSCAN analysis performed on overlapping fragments of the genes encoding part of ORF1 and the entire ORF2 and ORF3 led to the identification of five dominant immunoreactive areas, one located on ORF1 and four on ORF2. However, only some ORF2 peptides proved to be immunorelevant epitopes for virus type discrimination. The potential use of ORF2-derived antigens as diagnostic tools is demonstrated.

Immunocytological detection of isolated tumour cells in the bone marrow of malignant melanoma patients: a new method for the detection of minimal residual disease

Aims: Immunocytologically detected isolated tumour cells indicate a poor prognosis. This has been shown in breast, gastrointestinal and lung cancer, and might thereby help to indicate adjuvant therapy. Immunocytology has been proved to be a reliable technique and enables a phenotypic tumour cell characterization. We find this technique superior to molecular biological techniques such as reverse transcriptase polymerase chain reaction RT-PCR. So far, immunocytological studies have not been performed in malignant melanoma patients and our study aimed to establish this approach in melanoma patients.Methods : Twenty melanoma patients who underwent surgery for lymph-node metastasis using a radical lymphadenectomy were studied. Using the immunoperoxidase method, cytospins of bone marrow aspirates (1.5×106cells per patient) were stained with the monoclonal antibody HMB-45. Nineteen patients who were surgically treated but did not suffer from malignant melanoma were included as a control group. Results: Four of the 20 patients showed isolated tumour cells in the aspirate. Three of these patients had stage IV disease. One patient had a stage III tumour (1/7; 14.3%). One patient was classified as stage II and did not show tumour cells in the bone marrow. No staining cells were found in the control group (n=19). Conclusions: Our study demonstrates that the immunocytological approach can be used as a new technique to detect occult tumour cell dissemination in malignant melanoma patients and supports previous findings in carcinoma of the stomach, colon, pancreas and other tumours.

Minimal residual cancer

Introduction There is now a body of literature that establishes the prognostic value of detecting micrometastatic spread of tumor cells to the BM and lymph nodes in a large variety of malignancies. An increasing number of investigators have therefore employed immunocytological approaches (with or without tumor enrichment procedures) to detect individual disseminated neoplastic cells based on their phenotypic characteristics. More recently, the development of molecular techniques such as the PCR has given the potential for detection based on tumor-specific genomic changes. At this conference investigators, actively working in the growing field of micrometastasis research, discussed the current concepts, clinical and research implications of detecting micrometastatic cells, whether at diagnosis, prior to autologous stem-cell transplantation or after therapy. The conference was attended by more than 300 scientists from 20 different countries. As stated by the organizing committee, the meeting had three major goals: n Providing a platform for young scientists to present their research. n Determining the clinical relevance of micrometastatic cells in a variety of tumors. n Presenting modern developments in the field of the detection and treatment of micrometastases. The success of the first aim was achieved, as evidenced by the 100 high quality abstracts which were presented and discussed in the workshops and poster sessions. The five plenary sessions concentrated on: (1) The tumor biology of early micrometastasis; (2) Advanced methods for characterisation of cancer cells; (3) Clinical relevance of minimal residual detection (MRD) detection; (4) MRD in stem-cell transplantation; (5) Therapeutic strategies in MRD. Specialized areas of micrometastasis research were covered in the four workshops: (1) Methodological challenges of MRD detection; (2) Metastasis in head and neck tumors; (3) Staging and monitoring of MRD; (4) Novel therapies against residual tumor cells.

Detection of circulating melanoma cells by immunomagnetic cell sorting

We developed a cellular approach to the identification of circulating melanoma cells in peripheral blood using immunomagnetic cell sorting. One hundred seventy-eight blood samples from 129 melanoma patients and 30 samples from healthy persons and nonmelanoma patients were examined. After density gradient centrifugation the interphase was incubated with the mAb 9.2.27. Positive cells were labeled with magnetic microbeads and enriched by immunomagnetic cell sorting. Cells were stained using an alkaline phosphatase-antialkaline phosphatase assay and examined by light microscopy. In spiking experiments, melanoma cells seeded at a concentration of one melanoma cell per ml whole blood could be detected reliably with the assay. Circulating melanoma cells were not found in 30 controls examined, nor were 9.2.27-positive cells found in 41 patients with primary malignant melanoma. In patients with regional lymph node metastases and in patients with disseminated disease, circulating 9.2.27-positive cells could be detected in 3 out of 22 patients (13.6%) and 10 out of 66 patients (15.2%) examined. We present a sensitive and specific immunocytological approach to detect circulating melanoma cells in peripheral blood. The method is not suitable for early detection of metastases but is a valuable tool for further investigating biological characteristics of circulating melanoma cells.

Active proton and urea transport by amphibian skin.

Proton secretion in frog skin is mediated by an electrogenic H+ pump. Pharmacological and immunocytological approaches have identified this pump as belonging to the V-ATPase class. The key role of this V-ATPase in proton secretion (acid-base balance) and as a membrane energizer of other solute transport from very dilute solutions is outlined. It is shown that the frog skin constitutes a model of a V-ATPase-dependent Na+ transport mechanism applicable to other freshwater animals. On the other hand, attempts to implicate the V-ATPase in the active urea transport that develops through the skin of salt-adapted frogs have failed; the nature of the different urea transporters located on apical and basal epithelial cell membranes and those responsible for active urea reabsorption remain to be identified.

In barley leaf cells, jasmonates do not act as a signal during compatible or incompatible interactions with the powdery mildew fungus ( Erysiphe graminis f. sp. hordei)

Summary We have studied a possible function of jasmonates as mediators in the host-pathogen interaction of barley (Hordeum vulgare L.) with the powdery mildew fungus Egh (Erysiphe graminis f. sp. hordei). Previous findings from whole-leaf extracts demonstrated that (i) extracts from infected barley leaves did not contain enhanced levels of jasmonates, (ii) transcripts of jasmonate-inducible genes were not expressed upon infection, and (iii) exogenous application of jasmonates did not induce resistance to Egh (Kogel et al., 1995). Nevertheless, the question arises whether or not jasmonates are involved in the interaction of barley with the powdery mildew fungus at the local site of infection. Using an immunocytological approach the analysis of leaf cross-sections from a susceptible barley cultivar and its near-isogenic mlo5-resistant line revealed no accumulation of JIP-23, the most abundant jasmonate inducible protein, neither in epidermal cells attacked by the pathogen nor in adjacent mesophyll cells. As a positive control, cross-sections from methyl jasmonate-treated leaf segments showed a strong signal for JIP-23 accumulation. Because the presence of the jasmonate-inducible protein is highly indicative for an already low threshold level of endogenous jasmonate (Lehmann et al., 1995), the lack of JIP-23 accumulation at the sites of attempted fungal infection clearly demonstrates the absence of enhanced levels of jasmonates. This excludes even a local rise of jasmonate confined to those single cells penetrated (Mlo genotype) or attacked (mlo5 genotype) by the fungus.

New approaches to in situ detection of nucleic acids

The present paper reviews recent results obtained by different molecular biology-based, immunocytological approaches to the localization and identification of nucleic acids in sections of biological material. Examples of sensitive, high-resolution detection methods for RNA, DNA or specialized DNA regions are presented. Special emphasis is placed on the potential values and limitations of these new methods.

Ultrastructural localization of endogenous hormones and receptors in bone tissue: An immunocytological approach in frozen samples

In addition to their well-known mechanical role, bones are essential for the regulation of calcium phosphate metabolism. Most of the peptide and steroid hormones acting on bone tissue exert their cellular effects directly on bone cells after binding to cell receptors. A combined method using immunocytology on ultrathin frozen sections has been applied to bone tissue and made possible the ultrastructural localization of endogenous hormones and hormone receptors. There is immunocytological evidence, at the osteoblast level, for:(1) the presence of calcitonin, parathyroid hormone, growth hormone, 1,25 dihydroxyvitamin D 3, aldosterone, corticosterone and progesterone; (2) the existence of 1,25 dihydroxyvitamin D 3 and progesterone receptors; (3) the absence of estradiol and receptors, testosterone and gonadoliberin. Ultimate goals of future studies are to extend the ultrastructural localization of hormones at the osteoclast level, to asses the distribution of adhesive proteins and of their receptors in bone cells and matrix, and to use well-calcified human bone samples in order to evaluate better the role of therapeutic agents.

Cellular localization and cytochemical probing of resistance reactions to arbuscular mycorrhizal fungi in a ?locus a? myc? mutant of Pisum sativum L.

Pisum sativum L. myc− mutants which fail to form arbuscular mycorrhiza have recently been identified amongst nod− mutants (Duc et al., 1989, Plant Sci. 60, 215–222). The reason for this resistance to symbiotic fungi has been investigated in the case of a ‘locus a’ mutant (P2) inoculated with Glomus mosseae (Nicol. and Gerd.) Gerd, and Trappe. The fungal symbiont formed viable appressoria in contact with the root surface but its development was stopped at the root epidermis. Abundant material was deposited on the inner face of root cell walls adjacent to the appressoria in the P2 mutant, but not in the wild-genotype parent cultivar (Frisson) forming a symbiotic mycorrhizal infection. Fluorescence, histochemical, cytochemical and immunocytological approaches were used to characterize the paramural deposits in epidermal and hypodermal cells of the mutant. Strong fluorescence under blue light indicated the accumulation of phenolic compounds although polymers like lignin or suberin were not localized. Proteins and glycoproteins were homogeneously distributed within the paramural deposits. In the latter, the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) reaction for 1,4-polysaccharide detection showed a heterogeneous composition with electron-dense points surrounded by non-reactive material, but cytological tests for cellulose and pectin gave weak responses as compared to epidermal and hypodermal walls of the wild genotype. β-1,3-Glucans indicative of callose were detected by in-situ immunolocalization in the paramural deposits below appressoria on mutant roots, but not in walls of the wild genotype. Thus, appressorium formation by G. mosseae on roots of the ‘locus a’ P. sativum mutant elicits wall modifications usually associated with activation of defence responses to pathogens. It is proposed that this locus must be involved in a key event in symbiotic infection processes in P. sativum, and the possible role of complex regulatory interactions between symbiosis and defence genes in endomycorrhiza development is discussed.

Association of an aminoacyl-tRNA synthetase complex and of phenylalanyl-tRNA synthetase with the cytoskeletal framework fraction from mammalian cells

The intracellular distribution of several mammalian aminoacyl-tRNA synthetases was investigated by biochemical and immunocytological approaches. The fraction of aminoacyl-tRNA synthetases bound to the detergent-insoluble cytoskeletal framework obtained after extraction of NRK cells by 0.1 % Triton X-100 was estimated, by activity measurements, to about 80% for phenylalanyl-tRNA synthetase and 40% for the high-molecular-weight (HMW) complex containing the seven aminoacyl-tRNA synthetases specific for glutamic acid, isoleucine, leucine, methionine, glutamine, lysine, and arginine. This association was shown to be salt-dependent. The subcellular localization of these enzymes was examined using an immunocytoiogical approach. When cultured cells were fixed with paraformaldehyde and then permeabilized with Triton X-100, a fairly uniform cytoplasmic labelling was observed with antibodies directed to the aminoacyl-tRNA synthetase complex or to phenylalanyl-tRNA synthetase. By contrast, when cells were extracted with 0.1% Triton X-100 prior to fixation with paraformaldehyde, the staining patterns obtained with antibodies to aminoacyl-tRNA synthetases were very similar to that obtained with antibodies to rough endoplasmic reticulum, as assessed by single or double indirect immunofluorescence microscopy. These results suggest that free and bound forms of these aminoacyl-tRNA synthetases may coexist within the cell. In addition to cytoplasmic labelling, antibodies directed to phenylalanyl-tRNA synthetase stained the nucleus of rapidly growing cells. The possible significance of this finding is discussed.