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In order further to identify the prevalence of anti-receptor autoantibodies in the sera of patients with dilated cardiomyopathy (DCM), we attempted to detect autoantibodies against a series of G-protein-coupled cardiovascular receptors in a well-defined population of DCM patients from Japan. Peptides corresponding to the sequences of the second extracellular loops of the human beta 1 and beta 2 adrenoceptors, alpha 1 adrenoceptors, M2 muscarinic acetylcholine receptors and angiotensin II-1 (AT1) receptors were used as antigens in an enzyme immunoassay to screen the sera from patients with DCM (n = 28). Nine sera from patients with DCM (32%) and 2 sera from healthy subjects (9%) recognized the beta 1 adrenoceptor peptide. Ten sera from patients (36%) and 3 sera from healthy subjects (13%) recognized the M2 receptor peptide. Thirty-six per cent of the patients with autoantibody against the beta 1 adrenoceptor peptide. Ten sera from patients (36%) and 3 sera from healthy subjects (13%) recognized the M2 receptor peptide. Thirty-six per cent of the patients with autoantibody against the beta 1 adrenoceptor had autoantibody against the M2 receptor. However, no significantly high frequencies of autoantibodies against the beta 2 adrenoceptor, alpha 1 adrenoceptor and AT1 receptor were found in DCM patients. Our results demonstrate that a subgroup of patients with DCM have a specific spectrum of autoantibodies which are specifically directed against the second extracellular loops of the beta 1 adrenoceptors and M2 muscarinic receptors rather than other cardiovascular receptors.

ABSTRACT. The cellular responses of the V79 cell cycle to K-252a, a protein kinase inhibitor, were examined by flow cytometry (FCM) and by time-lapse videomicrography. At a concentration of 0.5 /M of K-252a for 72 h, V79 cells became hyperploid, having above 32c DNA content. The cycle times in the hyperploidizing process were ll.2 (4c-8c), 16.3 (8c-16c) and 19.3 (16c-32c) hours, values which were less than twice that of the control (10.5 h). After the washout of K-252a, the DNA content was widely distributed in the V79 cell population and hyperploid V79 cells reduced DNA content through a variety of cell division modes. These cellular responses to hyperploidization of V79 cells by K-252a were in reasonable agreement with those by demecolcine.The cell cycle is regulated by the phosphorylation of proteins controlling the cell cycle (5, 6). K-252a, a potent protein kinase inhibitor (10), has been recently reported to hyperploidize cultured cells (9). The mechanisms by which this drug causes hyperploidization, however, are slightly understood (1 , 9), and therefore it would be of interest to conduct an investigation on this. D emecolcine (colcemid) as well as colchicine prevent the formation of spindle fibers in the M phase (7, 8). We reported that V79, B16-F10 and Meth-A cell lines are hyperploidized by demecolcine at a wide range of concentrations, but thatHeLa-S3, L1210, k562 and B16 melanomacells were not (3). R ecently, a new model, in which the cell cycle consists of two-linked cycles, DNA replication and cell division, has been presented (2, ll). We interpreted our results based on this model wherein the cell lines hyperploidized by demecolcine generate the start signals for the DNA cycle before the drug affects the cell division cycle (3).

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ABSTRACT. Demecolcine (Colcemid), an inhibitor of spindle fiber formation in M phase, induced apoptosis in V79 cells. At a concentration of 0.01^g/ml demecolcine, V79 cells proliferated exponentially as well as controls, although temporal Mphase accumulation occurred 6 h after the addition of demecolcine. At 0.1 /*g/ml, the cells became hyperploid after remaining in the Mphase for some time. Apoptosis occurred in V79 cells exposed to demecolcine at a concentration of 0.03 /ug/m\. Apoptosis was defined as the appearance of a sub-Gl peak in DNAhistograms and a ladder pattern of fragmented DNAin gelelectrophoresis. Demecolcine (Colcemid) as well as colchicine prevents the formation of spindle fibers in M phase (8, 9 Apoptosis is a mode of cell death that involves the active participation of the cell in its own destruction and is distinct from necrosis. The general characteristics of apoptosis are well established. They consist of distinct morphological and biochemical changes that include cell shrinkage, cell surface blebbing, segmentation of the nucleus and extensive degradation of DNAinto oligonucleosomal-sized fragments (6, 10).Oneof the most commonmeansof detecting apoptosis is DNAgel electrophoresis, in which DNAfragments can be seen as a "ladder" of bands equivalent to multiples of mononucleosomes or oligonucleosomes. Flow cytometry (FCM)can identify apoptotic cells as a subdiploid peak, after staining with DNAspecific dyes (1,12). MATERIALS AND METHODCells. V79 cells (Chinese hamster lung cell line) were maintained in a humidified atmosphere of 5%CO2at 37°C as a monolayer culture in a Leibovitz's L15 : Ham's F10 mixture (7 : 3) supplemented with 10% newborn calf serum (M.A. Bioproducts), streptomycin (100 //g/ml) and penicillin (50 units /ml). The cells were cultured at low densities.Drug treatment. Exponentially growing V79 cells were plated in Petri dishes (60 mmdiameter, NUNC)at a density of about 1 x 105 cells/dish, then the medium was changed 24 h after seeding. Twelve hours thereafter, the cells were exposed to demecolcine (Sigma Chemical Co.) at various concentrations. At 6, 12, 24 and 48 hours thereafter, the cells were harvested and prepared for flow cytometry, cell growth measurements, nuclear morphology, and DNAgel-electrophoresis. CellpreparationforFCM. At various times after the drug addition, the V79 cells were washed twice with PBS(~> (divalent cation-free phosphate-buffered saline) and trypsinized (0.17% trypsin and 30 mMEDTA). The cells were fixed with 20% ethanol, then incubated with 0.25% RNase (Type II-A, Sigma Chemical Co.) for 3 h at 4°C. Immediately before measurements, the cells were stained with PI (propidium iodide, 7.5 x 10~5 M) and red fluorescence was examined by means of FCM.Under these staining conditions, the signal due to residual double stranded RNAis negligible and the relative intensity of red fluorescence corresponds to the DNAcontent (7). Flow cytometry. Fluorescence from individual cells was measured using a Cytofluorograf system 50H (Ortho Instruments) equipped with ...