Adult rabbit cardiomyocytes undergo typical hibernation-like dedifferentiation when co-cultured with cardiac fibroblasts.

Objectives: Little is known about the causal factors which induce the typical structural changes accompanying cardiomyocyte dedifferentiation in vivo such as in chronic hibernating myocardium. For identifying important factors involved in cardiomyocyte dedifferentiation, as seen in chronic hibernation, an in vitro model mimicking those morphological changes, would be extremely helpful. Methods: Adult rabbit cardiomyocytes were co-cultured with cardiac fibroblasts. The typical changes induced by this culturing paradigm were investigated using morphometry, electron microscopy and immunocytochemical analysis of several structural proteins, which were used as dedifferentiation markers, i.e., titin, desmin, cardiotin and alpha -smooth muscle actin. Results: Close apposition of fibroblasts with adult rabbit cardiomyocytes induced hibernation-like dedifferentiation, similar to the typical changes seen in chronic hibernation in vivo. Both changes in ultrastructure and in the protein expression pattern of dedifferentiation markers as seen in chronic hibernating myocardium were seen in the co-cultured cardiomyocytes. Conclusion: Hibernation-like changes can be induced by co-culturing adult rabbit cardiomyocytes with fibroblasts. This cellular model can be a valuable tool in identifying and characterizing the pathways involved in the dedifferentiation phenotype in vivo, and already suggests that many of the structural changes accompanying dedifferentiation are not per se dependent on a decreased oxygen availability

Both lamin A and lamin C mutations cause lamina instability as well as loss of internal nuclear lamin organization.

We have applied the fluorescence loss of intensity after photobleaching (FLIP) technique to study the molecular dynamics and organization of nuclear lamin proteins in cell lines stably transfected with green fluorescent protein (GFP)-tagged A-type lamin cDNA. Normal lamin A and C proteins show abundant decoration of the inner layer of the nuclear membrane, the nuclear lamina, and a generally diffuse localization in the nuclear interior. Bleaching studies revealed that, while the GFP-tagged lamins in the lamina were virtually immobile, the intranuclear fraction of these molecules was partially mobile. Intranuclear lamin C was significantly more mobile than intranuclear lamina A. In search of a structural cause for the variety of inherited diseases caused by A-type lamin mutations, we have studied the molecular organization of GFP-tagged lamin A and lamin C mutants R453W and R386K, found in Emery–Dreifuss muscular dystrophy (EDMD), and lamin A and lamin C mutant R482W, found in patients with Dunnigan-type familial partial lipodystrophy (FPLD). In all mutants, a prominent increase in lamin mobility was observed, indicating loss of structural stability of lamin polymers, both at the perinuclear lamina and in the intranuclear lamin organization. While the lamin rod domain mutant showed overall increased mobility, the tail domain mutants showed mainly intranuclear destabilization, possibly as a result of loss of interaction with chromatin. Decreased stability of lamin mutant polymers was confirmed by flow cytometric analyses and immunoblotting of nuclear extracts. Our findings suggest a loss of function of A-type lamin mutant proteins in the organization of intranuclear chromatin and predict the loss of gene regulatory function in laminopathies

A novel flow cytometric steorid hormone receptor assay for paraffin embedded breast carcinomas : An objective quantification of the steroid hormone receptors and direct correlation to ploidy status and proliferative capacity in a single-tube assay.

Semiquantitative estimation of steroid hormone receptors by immunohistochemistry applied to paraffin sections is common practice in surgical pathology Flow cytometric (FCM) analysis of estrogen receptor (ER) and progesterone receptor (PR) levels provides a faster and more objective quantitative assay. However, a major problem in such FCM analyses of solid tumor samples is the admixture of tumor cells with normal epithelial, stromal, and inflammatory cells. The aim of the underlying study was to investigate the applicability of a recently developed multiparameter flow cytometric methodology for the accurate estimation of the fraction of steroid hormone receptor-positive tumor cells and to explore whether this multiparameter approach allows the detection of specific, clinically relevant subsets of tumors, based on a combination of ploidy level, steroid hormone receptor status, and cell cycle characteristics. For this purpose, samples of 42 breast cancer patients, from which routine immunohis tochemistry for ER and PR also was available, were analyzed. From each case, a cell suspension was prepared from the paraffin block by applying a heating and short pepsin digestion step to 50-mu m-thick sections. These cell suspensions were double-immunostained for cytokeratin to identify the epithelial cells, and ER or PR, whereas DNA was quantitatively stained with propidium iodide using an optimized protocol. In the entire group of breast tumors, the percentages of ER- and PR-positive cells were registrated in the epithelial subfraction, in combination with DNA ploidy and S phase fraction (SPF). A significant correlation was found between the fraction of hormone receptor-positive cells as found by the immunohistochemical and FCM procedures. For ER, a correlation coefficient of r = .87 was found, and for PR r = .62, both P <.0001. It became dear that all the diploid breast tumors had more than 30% tumor cells positive for ER with a SPF lower than 10%, whereas aneuploid tumors contained on average a smaller percentage of steroid hormone receptor-positive cells, and simultaneously an SPF greater than 10%. Our results show that this multiparameter FCM analysis allows an objective and reproducible quantification of the fraction of steroid hormone receptor-positive cells in the relevant epithelial cell compartment in relation to DNA ploidy status and proliferative capacity in a single-tube assay. by W.B. Saunders Company

S-nitroso-N-acetylpenicillamine and nitroprusside induce apoptosis in a neuronal cell line by the production of different reactive molecules.

CHP212 neuroblastoma cells were exposed to two different nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside. Apoptosis and necrosis were determined with flow cytometric analysis of annexin V binding and propodium iodide uptake. Both S-nitroso-N-acetylpenicillamine and sodium nitroprusside induced apoptosis, but with a different time dependency. Oxyhemoglobin (NO scavenger) attenuated the toxicity of S-nitroso-N-acetylpenicillamine, but had no effect on the toxicity of sodium nitroprusside. By contrast, deferoxamine (iron chelator) attenuated the toxicity of sodium nitroprusside, but had no effect on the toxicity of S-nitroso-N-acetylpenicillamine. Urate (ONOO- scavenger) did not influence the toxicity of either S-nitroso-N-acetylpenicillamine or sodium nitroprusside, but protected from SIN-1 (3-morpholinosydnonimine, ONOO- donor). It was shown that both dithiothreitol and ascorbic acid affected the toxicity of S-nitroso-N-acetylpenicillamine and sodium nitroprusside in opposite ways. In the presence of dithiothreitol, superoxide dismutase and catalase decreased the toxicity of sodium nitroprusside. In the presence of cells, but not in their absence, S-nitroso-N-acetylpenicillamine decomposed with a half-life of about 4 h as assessed by the production of nitrite and absorbance reduction at 335 nm. Sodium nitroprusside decomposed Very slowly in the presence of cells as assessed by the production of ferrocyanide. It can be concluded that (1) slow and sustained release of NO from S-nitroso-N-acetylpenicillamine at the cell surface causes apoptosis in CHP212 cells, probably without the involvement of ONOO-, (2) sodium nitroprusside causes apoptosis by the production of H2O2 and/or iron, rather than NO, and probably has to be taken up by the cell for decomposition

Clonality assessment of lymphoproliferative disorders by multiparameter flow cytometry of paraffin-embedded tissue : an additional diagnostic tool in surgical pathology.

A major drawback of immunohistochemical detection of monoclonality in B-cell lymphoproliferative disorders is the lack of contrast between surface-immunoglobulin staining and extracellular immunoglobulin staining. To bypass this drawback, immunophenotyping of single-cell suspensions by flow cytometry is commonly used. Although the expression of immunoglobulin light chain subtype can be quantified rapidly and reliably, the technique is hampered by the requirement of fresh unfixed material. We applied a recently developed technique for the isolation of single cells from formalin-fixed, paraffin-embedded material to measure clonality in B-cell lymphoproliferative disorders (lymphoid tissue (n = 10) and non-Hodgkin's B-cell lymphoma (n = 10). Immunocytochemistry indicated that common cell surface markers as well as the immunoglobulin light chains could be detected in the cell suspensions derived from archival material. In addition, the technique also allowed combined high-resolution DNA flow cytometric analysis. To investigate the effect of formalin fixation on cross-linking of extracellular immunoglobulins to lymphocytes, a double-immunostaining experiment for both light chain immunoglobulins (kappa and lambda) was performed. This experiment showed that this crosslinking was minimal (less than 2%). All cases of reactive lymphoid hyperplasia were DNA diploid and showed a polyclonal expression of immunoglobulin light chains. In contrast, in 9 of 10 non-Hodgkin's B-cell lymphomas, monoclonality was established on the basis of light chain expression, whereas only 6 of 9 cases were conclusive by immunohistochemistry. Four of the 9 cases were DNA aneuploid. One case did not show light chain expression at all by both techniques. However, this case could be classified as malignant by flow cytometric analysis because of the DNA-aneuploid nature of the B-cell subpopulation. The average S-phase fraction (SPF) of the B cells in the reactive lymphoid tissues was 3.5%. The mean SPF values for B cells in DNA-diploid cases of lymphomas was 3.0%, whereas the mean SPF of B cells in DNA-aneuploid cases was 6.1%. The presented technique is superior to immunohistochemistry for the detection of monoclonality in B-cell lymphoproliferative disorders and therefore provides a powerful tool to support the diagnosis of malignant lymphoma in routinely processed archival samples of lymphoid tissues. HUM PATHOL 31:422-427. by W.B. Saunders Company

Localization of smoothelin in avian smooth muscle and identification of a vascular-specific isoform

AbstractSmoothelin is a smooth muscle-specific protein of minor abundance first identified via a monoclonal antibody obtained using an avian gizzard extract as antigen. Dual labelling of ultrathin sections with antibodies to smoothelin together with antibodies to other smooth muscle proteins showed that smoothelin was co-distributed with filamin and desmin in the cytoskeleton domain of the smooth muscle cell. From the finding that smoothelin, unlike desmin, was readily extracted by Triton X-100 as well as under conditions that solubilized myosin, β-actin and filamin, we conclude that smoothelin is most likely associated with the actin cytoskeleton. Western blot analysis of gizzard smooth muscle tissue revealed an immunoreactive protein band with an apparent molecular weight of 59 kDa that separated into 3–4 isolated variants, while avian vascular muscle showed a polypeptide band of 95 kDa. These results point to the presence of specific isoforms in visceral and vascular smooth muscles. The 59 kDa isoform was shown to be distinct from the 60 kDa filamin-binding protein, described by Maekawa and Sakai (FEBS Lett. 221, 68–72, 1987). As compared to other smooth muscle markers, such as calponin and SM22, smoothelin appeared very late during differentiation in the chick gizzard, on about the 18th embryonic day