Sexual behaviour in Euplotes raikovi is accompanied by pheromone-induced modifications of ionic currents.

In the marine ciliate Euplotes raikovi, pheromone released by a complementary mating type (nonself pheromone) induces typical sexual behaviour, whereas self pheromone released by the same mating type generally has no effect. Nonself pheromone evokes a reduction of the mean walking speed by 66 %, a threefold increase in the frequency and duration of long-lasting rest phases and a doubling in the number of side-stepping reactions. Consequently, translocation is strongly reduced and the cells remain in a small area. This could increase the probability of finding a sexual partner for pair formation (conjugation). The usual pattern of rhythmic, spontaneous depolarizations controlling the walking rhythm is absent in nonself-pheromone-stimulated cells. The remaining depolarizations arise from a 4 mV hyperpolarized membrane potential and do not reach the usual amplitudes of 15-20 mV but only of 6-10 mV. In addition, the amplitudes of K+ currents are increased at depolarizations of more than 20 mV by at least 30 %. Hyperpolarization- and depolarization-activated Na+ current amplitudes are increased, whereas the Ca2+ current amplitude remains nearly unaffected.

An immunosensor based on disposable electrodes for rapid estimation of fatty acid-binding protein, an early marker of myocardial infarction.

An immunosensor was developed that allows the rapid estimation of fatty acid-binding protein (FABP) in neat plasma samples. FABP is released into the blood following myocardial infarction and elevated levels are found already 3 h after onset of symptoms. The sensor is based on screen-printed graphite working and Ag/AgCl reference electrodes and an immunosandwich procedure for the quantification of FABP. The capture antibodies are bound to the electrode surface by adsorption and will trap FABP from the plasma sample. The sandwich is then completed by a second monoclonal antibody conjugated with alkaline phosphatase. The enzyme converts p-aminophenylphosphate to p-aminophenol, which is detected amperometrically at +350 mV. The high binding capacity and very short response time of the working electrode allow within 20 min the quantification of FABP in the measuring range 10-350 ng/ml, covering the pathological range of FABP release into the circulation. Measurements of plasma samples from a patient with acute myocardial infarction show an excellent correlation of the results obtained with the biosensor and those obtained with the respective reference ELISA. Owing to the long stability of the electrodes with immobilized capture antibody (> 3 months) a quick application without the need of labour-intensive electrode preparation is possible.

Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins.

A decade of studies on the human nuclear antigen defined by monoclonal antibody Ki-67 (the "Ki-67 protein") has made it abundantly clear that this structure is strictly associated with human cell proliferation and that the expression of this protein can be used to assess the growth fraction of a given cell population. Until recently the Ki-67 protein was described as a nonhistone protein that is highly susceptible to protease treatment. We have isolated and sequenced cDNAs encoding for this antigen and found two isoforms of the full length cDNA of 11.5 and 12.5 kb, respectively, sequence and structure of which are thus far unique. The gene encoding the Ki-67 protein is organized in 15 exons and is localized on chromosome 10. The center of this gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp ("Ki-67 repeats"), each containing a highly conserved new motif of 66 bp ("Ki-67 motif"). The deduced peptide sequence of this central exon possess 10 ProGluSerThr (PEST) motifs which are associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors, etc. Like the latter proteins the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit 3H-thymidine incorporation in permanent human tumor cell lines in a dose-dependent manner.

Assessment of cell proliferation by means of an enzyme-linked immunosorbent assay based on the detection of the Ki-67 protein.

A new ELISA system for the estimation of cell proliferation based on the detection of the Ki-67 protein is described. This protein has turned out to be strictly correlated to all active parts of the cell cycle, i.e., G1, S, G2, and mitosis, but is absent in G0. In addition, it is not detectable during DNA repair. In cultures of cell line cells as well as stimulated peripheral blood cells the values obtained with this ELISA system paralleled the [3H]thymidine uptake in different cell cultures. Thus, this assay provides a simple, non-radioactive assessment of proliferation of cultured cells.

Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein).

AIMS--To elucidate the fine specificities of the antibodies MIB 1 and MIB 3 and of additional monoclonal antibodies which also recognise the Ki-67 protein (MIB 5, IND.64, JG-67-2a). METHODS--Different parts of the Ki-67 protein cDNA were expressed in Escherichia coli. Bacterial lysates were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on to nitrocellulose. Additionally different peptides were synthesised on a membrane support (SPOT-Blot). The immunoreactivity of the antibodies with the recombinant proteins and the immobilised synthetic peptides, respectively, was analysed. A competition enzyme linked immunosorbent assay (ELISA) using a soluble synthetic peptide was also performed. RESULTS--The epitopes of all antibodies tested were contained within the same region of seven amino acids. The antibodies MIB 1 and MIB 3 required the five amino acid sequence FKELF for binding, whereas Ki-67, JG-67-2a, MIB 5 and IND.64 detected the sequence FKEL. CONCLUSIONS--It is concluded that the amino acid sequence FKELF represents an immunodominant area of the Ki-67 protein and that there is no correlation between the ability to detect the Ki-67 protein in paraffin wax sections irradiated with microwaves and the epitopes recognised by the antibodies.

New antiserum against Ki-67 antigen suitable for double immunostaining of paraffin wax sections.

AIMS: To prepare a rabbit antiserum equivalent to MIB 1 to permit the simultaneous assessment of cell proliferation and other markers of interest using double labelling studies. METHODS: Rabbits were immunised with a synthetic peptide deduced from the cDNA sequence coding for the Ki-67 antigen. Serum samples were tested for immunoreactivity using different immunobiochemical methods. RESULTS: A polyclonal antiserum was derived which detects the native as well as recombinant parts of the Ki-67 antigen in different test systems. Furthermore, the antiserum stains the Ki-67 antigen in routinely processed, paraffin wax embedded material. CONCLUSIONS: After antigen unmasking by microwave treatment the antiserum described here represents a powerful tool for the determination of growth fractions even in archival material. It is especially suitable for double staining experiments in combination with monoclonal antibodies.

The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins.

The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length cDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated cDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen cDNA. The central part of the Ki-67 antigen cDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycle-associated nuclear nonhistone proteins.

Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections.

Enzymatic and non-enzymatic treatments for antigen unmasking on formalin-fixed, paraffin-embedded, dewaxed sections were optimized and compared by the use of a panel of antibodies of diagnostic relevance (anti-cytokeratins, vimentin, S-100, T- and B-cell receptors, Ki-67/MIB 1, muscle actin). Non-enzymatic unmasking was obtained by boiling the slides in a microwave oven in 0.01 M salt solution (pH 6) or in 6 M urea. Trypsin or pronase digestion was used for comparison and found to be necessary for some of the reagents. The investigation was then extended to 256 antibodies; the epitopic amino acid sequence was known for 48 of them. We found that enzymatic and non-enzymatic antigen unmasking are not dependent on the epitope sequence, but some antigens benefit selectively from one treatment but not from the other. Denaturation of proteins is the likely mechanism which leads to immunodetection on microwave oven-boiled slides; this suggestion is supported by the use of denaturating solutions and by the observation that endogenous enzymes were inactivated and a few antigens were no longer immunodetectable after boiling. Non-enzymatic methods for antigen unmasking are a powerful new tool for broadening the use of antibodies for immunostaining formalin-fixed, paraffin-embedded sections and should be used in parallel with the traditional enzymatic methods.

New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope.

BACKGROUND: The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all cells that are not in G0. Recently, we could demonstrate that Ki-67 detects a double band in Western blots of proliferating cells with apparent molecular weights of 345 kilodaltons and 395 kilodaltons, respectively. Furthermore, initial molecular biologic data favored the view that the epitope detected by Ki-67 might be encoded by a repetitive 66 bp element. EXPERIMENTAL DESIGN: In order to verify this assumption, parts of the Ki-67 cDNA were bacterially expressed, and the fusion proteins obtained were used to elicit new monoclonal antibodies. The specificities of the new reagents were tested by immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay techniques. RESULTS: The somatic cell fusions revealed a number of antibodies with immunoreactivities comparable to Ki-67. Three antibodies, designated MIB 1-3, were further characterized. Besides the fact that their immunostaining reactivity is identical with that of Ki-67, all new antibodies react in Western blots with native Ki-67 antigen. Furthermore, Western blot and competitive binding assays by enzyme-linked immunosorbent assay clearly demonstrate that MIB 1 and MIB 3, like the original Ki-67 antibody, react with an epitope that is encoded by the 66 bp repetitive element mentioned above. MIB 2, however, reacts with an epitope distinct from this latter structure. In addition, after antigen unmasking by microwave treatment, MIB 1 and MIB 3 detect the Ki-67 antigen in paraffin sections. CONCLUSIONS: Our results demonstrate that it is possible to use bacterially expressed parts of the Ki-67 antigen as immunogen to elicit antibodies that react with the native antigen. While MIB 1 and MIB 3 detect the same or a very similar epitope as the original antibody Ki-67, MIB 2 clearly differs in its fine specificity. Our results provide a circle of evidence that the cDNA sequence thus far determined encodes for the Ki-67 antigen. Furthermore, the new antibodies may become powerful tools for routine histopathology and for further functional characterization of the Ki-67 antigen.

Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections.

The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all active parts of the cell cycle. Recently we have raised monoclonal antibodies, MIB 1-3, against recombinant parts of the Ki-67 antigen. These antibodies are true Ki-67 equivalents, as demonstrated by immunostaining of fresh specimens, biochemistry, and molecular biological techniques. Formalin-fixed, paraffin-embedded sections routinely processed for immunohistochemistry failed to stain for Ki-67 and MIB 2. Antibodies MIB 1 and MIB 3 labelled mitotic figures, while non-mitotic proliferating cells were negative under these conditions. However, when dewaxed microwave oven-processed paraffin sections of formalin-fixed tissues were used, MIB 1 and MIB 3 gave strong nuclear staining of those cells presumed to proliferate under a variety of normal and neoplastic conditions. Moreover, routine decalcification or depigmentation techniques did not alter the immunoreactivity of MIB 1 and MIB 3 with microwave-processed paraffin sections. This method is highly reproducible, easy to perform at low cost, and no additional technical skill is needed because after microwave treatment just routine immunohistochemical methods are used. Since we have successfully applied this new method to sections obtained from paraffin blocks stored for a long time (in one case more than 60 years), the assessment of cell kinetics through the detection of Ki-67 antigen is now possible on archival material collected in histopathology departments all over the world.

Monoclonal antibody JC1: new reagent for studying cell proliferation.

AIM: To characterise a newly developed mouse monoclonal antibody JC1 which recognises a nuclear antigen present in proliferating cells in normal tissues and neoplastic lesions, and which is absent in resting cells. METHODS: The methodology was established using a representative range of frozen sections from normal tissues and from certain tumours which were immunostained with antibodies Ki67 and JC1. The molecular weight of the antigen recognised by JC1 was obtained by western blot analysis and this was compared with that of Ki67. IM-9 cell lysates containing Ki67 derived plasmids were also tested with JC1 antibody. RESULTS: Biochemical investigation indicated that the antigen recognised by JC1 gives two molecular weight bands of 212 and 123 kilodaltons, which is distinct from the well characterised anti-proliferation monoclonal antibody Ki67 (395-345 kilodaltons). In addition recombinant Ki67 protein is not recognised by JC1. Immunohistological reactivity was seen in areas known to contain numerous proliferating cells such as lymphoid germinal centres, splenic white matter, cortical thymocytes and undifferentiated spermatogonia. In tumours many cells from adenocarcinomas, oat cell carcinomas, squamous cell carcinomas of lung, and seminomas were labelled by JC1 with a distribution and proportion similar to that seen with Ki67. In normal tissues the only apparent difference was in testis where JC1 stained a considerably greater number of cells than Ki67. In all cases studied the new antibody showed nuclear reactivity only. JC1 did not show any cytoplasmic crossreactivity with squamous cells as is frequently seen with Ki67. CONCLUSION: Antibody JC1, which recognises a nuclear antigen present in proliferating cells, should provide a useful adjunct to Ki67 as a marker of proliferation especially in those cases such as squamous cell carcinomas where a Ki67 index cannot be determined.

Production of a monoclonal antibody (IND.64) identifying a cell cycle-associated antigen using spleen cells from nude mice bearing Ichikawa tumour.

Using spleen cells from athymic nude mice grafted with Ichikawa tumour, we have generated the monoclonal antibody IND.64, which detects a proliferation-associated nuclear antigen. Immunoblotting analysis with IND.64 showed a double band with apparent molecular weights of 395 and 345 kD. In normal human tissues, the antigen detected by IND.64 was expressed only by the nuclei of proliferating cells, such as germinal centre cells of reactive lymph nodes, cortical thymocytes, the basal layer of the skin, and proliferative compartments of the stomach, small intestine, and colon. IND.64 did not react with cells known to be non-proliferative or to show only a low turnover, such as cells of the kidney, liver, smooth muscle, cardiac muscle, and brain. The expression of this antigen during the cell cycle was determined using two approaches: IND.64 immunostaining of synchronized adult bovine aortic endothelial cells and flow cytometric analysis of double-labelled PHA-stimulated peripheral mononuclear blood leucocytes with a DNA marker and IND.64. The antigen recognized by IND.64 was found to appear in the late G1 phase, and persisted in phases S, G2, and M, but was absent in the G0 and early G1 phases. IND.64 was further investigated in different tumour types to evaluate the correlation between the percentage of IND.64-positive cells (IND.64 index) and the histological grade. In non-Hodgkin's lymphomas, an excellent correlation was found between the percentage of IND.64-positive cells and the cytomorphological grade. In nodular sclerosis and mixed cellularity Hodgkin's disease, a high number of Reed-Sternberg cells were positive with IND.64. The non-lymphoid neoplasms investigated showed a variable percentage of positive cells. IND.64 appears to be a promising tissue marker to complement the evaluation of prognosis in human cancer.

Immunobiochemical characterization of the antigen detected by monoclonal antibody IND.64. Evidence that IND.64 reacts with the cell proliferation associated nuclear antigen previously defined by Ki-67.

The immunohistochemical characteristics of the monoclonal antibody IND.64 are very similar to those of the monoclonal antibody Ki-67. The aim of this study was to further characterize this new antibody and to compare it with Ki-67 using immunobiochemical methods. Our results demonstrate that the similarity between the antibodies holds true even at the molecular level. Immunoblot analysis of IM-9-cell lysates with both antibodies showed a double band with apparent molecular weights of 395 kD and 345 kD, respectively. Competition ELISAs using a synthetic peptide derived from the thus far determined Ki-67 cDNA sequence as competitor, indicate that IND.64 may recognize the same epitope as Ki-67. The IND.64 epitope resides at least within a 20 amino acid sequence which also contains the Ki-67 epitope. Since IND.64 is of the IgG2b subclass, while Ki-67 is of the IgG1 subclass, the two antibodies may be useful for double immunostaining. In addition, IND.64 may help in determining the still unknown function of the antigen it recognizes.

The St Croix disaster and the National Disaster Medical System.

The National Disaster Medical System was designed to respond to a catastrophic disaster by creating a group of specially trained civilian disaster medical assistance teams. The teams would be transported to the periphery of the event to triage, stabilize, and then prepare victims for evacuation to facilities elsewhere in the United States that have agreed in advance to accept such patients. Hurricane Hugo's devastation in St Croix offered the first opportunity to test the system. The event was an example of a type of medical disaster that resulted in a sudden reduction in medical resources without a great increase in casualties. Background information and operation of the New Mexico disaster medical assistance team are presented with a clinical profile of the patients seen during the disaster. We describe the first actual deployment of a disaster medical assistance team and the issues that must be addressed before future deployments.

Monoclonal antibodies identify common and differentiation-specific antigens on the plasma membrane of guard cells of Vicia faba L.

Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.