Breast cancer carcinoma-associated fibroblasts differ from breast fibroblasts in immunological and extracellular matrix regulating pathways.

Tumor stroma has been recently shown to play a crucial role in the development of breast cancer. Since the origin of the stromal cells in the tumor is unknown, we have examined differences and similarities between three stromal cell types of mesenchymal origin, namely carcinoma associated fibroblasts from breast tumor (CAFs), fibroblasts from normal breast area (NFs) and bone marrow derived mesenchymal stromal cells (MSCs). In a microarray analysis, immunological, developmental and extracellular matrix -related pathways were over-represented in CAFs when compared to NFs (p<0.001). Under hypoxic conditions, the expression levels of pyruvate dehydrogenase kinase-1 (PDK1) and pyruvate dehydrogenase kinase-4 (PDK4) were lower in CAFs when compared to NFs (fold changes 0.6 and 0.4, respectively). In normoxia, when compared to NFs, CAFs displayed increased expression of glucose transporter 1 (GLUT-1) and PDK1 (fold changes 1.5 and 1.3, respectively). With respect to the assessed surface markers, only CD105 was expressed differently in MSCs when compared to fibroblasts, being more often expressed on MSCs. Cells with myofibroblast features were present in both NF and CAF samples. We conclude, that CAFs differ distinctly from NFs at the gene expression level, this hypothesis was also tested in silico for other available gene expression data.

Image-based assessment of microvascular function and structure in collagen XV- and XVIII-deficient mice.

Collagen XV and XVIII are ubiquitous constituents of basement membranes. We aimed to study the physiological roles of these two components of the permeability barrier non-invasively in striated muscle in mice deficient in collagen XV or XVIII by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Structural information was obtained with transmission electron microscopy (TEM). MR data were analysed by two different analysis methods to quantify tissue perfusion and microcirculatory exchange parameters to rule out data analysis method-dependent results. Control mice (C57BL/6J Ola/Hsd strain) or mice lacking either collagen XV (Col15a1(-/-)) or XVIII (Col18a1(-/-)) were included in the study. MR images were acquired using a preclinical system using gadodiamide (Gd-DTPA-BMA, molecular weight 0.58 kDa) as a tracer. Exchange capacity (permeability (P)-surface area (S) product relative to blood flow (FB)) was increased in test mice compared to controls, but the contributions from P, S, and FB were different in these two phenotypes. FB was significantly increased in Col18a1(-/-), but slightly decreased in Col15a1(-/-). PS was significantly increased only in Col18a1(-/-) even though P was increased in both phenotypes suggesting S might also be reduced in Col15a1(-/-) mice. Immunohistochemistry and electron microscopy demonstrated alterations in capillary density and morphology in both knockout mouse strains in comparison to the control mice. Both collagen XV and XVIII are important for maintaining normal capillary permeability in the striated muscle. DCE-MRI and the perfusion analyses successfully determined microvascular haemodynamic parameters of genetically modified mice and gave results consistent with more invasive methods.

Alcohol-induced premature permeability in mouse placenta-yolk sac barriers in vivo.

OBJECTIVE: Acute alcohol exposure induces malformation and malfunction of placenta-yolk sac tissues in rodents, reducing the labyrinth zone in the placenta and altering the permeability and fluidity of the cell membrane. During normal mouse placentation the cells line up in an optimal way to form a hemotrichorial placenta where layers II and III are connected through gap junctions. These act as molecular sieves that limit the passage of large molecules. PlGF is a developmentally regulated protein that controls the passage of molecules in the vasculosyncytial membranes and media of large blood vessels in the placental villi. In addition to the chorioallontoic placenta, rodents also have another type of placenta that consists of Reichert's membrane within the trophoblast cell layer on the maternal side and the parietal endodermal cells on the embryonic site. This forms a separate materno-fetal transport system. We study here whether alcohol affects these two placental barriers, leading to placental malfunction that in turn diminishes the nutrient supply to the embryo. STUDY DESIGN: CD-1 mice received two intraperitoneal injections of 3 g/kg ethanol at 4 h intervals at 8.75 days post coitum (dpc). The placentas were collected on 9.5, 11.5 and 14.5 dpc and used for histopathological protein studies. Hemotrichorial cell layer structure interactions through connective tissue and gap junction were analyzed by electron microscopy. The permeability of the feto-maternal barrier was visualized with Evans Blue. RESULTS: VEGF, a permeability inducer, was found to be up-regulated in the mouse placenta after acute alcohol exposure, and permeability was also affected by altered structures in the barriers that separate the feto-maternal blood circulation which destroyed the gap junctions in the hemotrichorial cell layer, reduced the thickness of Reichert's membrane and interfered with with Reichert's trophoblast/Reichert's parietal interaction. These defects together could have caused the permeability malfunction of the placenta-yolk sac tissues as visualized and quantified here by Evans Blue leakage. CONCLUSIONS: An altered PlGF/VEGF ratio together with barrier malformation may contribute to placental malfunction by altering the permeability of the feto-maternal barriers. Further studies are needed in order to show whether premature permeability is involved in the intrauterine growth restriction observed in human FAS embryos.

Birch pollen allergen Bet v 1 binds to and is transported through conjunctival epithelium in allergic patients.

BACKGROUND: Previous work in type-I pollen allergies has mainly focused on lymphocytes and immune responses. Here, we begin to analyse with a systems biology view the differences in conjunctival epithelium obtained from healthy and allergic subjects. METHODS: Transcriptomics analysis combined with light and electron microscopic analysis of birch pollen allergen Bet v 1 located within conjunctival epithelial cells and tissues from birch allergic subjects and healthy controls was carried out. RESULTS: Bet v 1 pollen allergen bound to conjunctival epithelial cells within minutes after the exposure even during the nonsymptomatic winter season only in allergic, but not in healthy individuals. Light- and electron microscopy showed that Bet v 1 was transported through the epithelium within lipid rafts/caveolae and reached mast cells only in allergic patients, but not in healthy individuals. Transcriptomics yielded 22 putative receptors expressed at higher levels in allergic epithelium compared with healthy specimens. A literature search indicated that out of these receptors, eight (i.e. 37%) were associated with lipid rafts/caveolae, which suggested again that Bet v 1 transport is lipid raft/caveola-dependent. CONCLUSIONS: We show a clear difference in the binding and uptake of Bet v 1 allergen by conjunctival epithelial cells in allergic vs healthy subjects and several putative lipid raft/caveolar receptors were identified, which could mediate or be co-transported with this entry. The application of discovery driven methodologies on human conjunctival epithelial cells and tissues can provide new hypotheses worth a further analysis to the molecular mechanisms of a complex multifactorial disease such as type-I birch pollen allergy.

Extracellular superoxide dismutase has a highly specific localization in idiopathic pulmonary fibrosis/usual interstitial pneumonia.

AIMS: Recent studies suggest the importance of oxidant stress in the progression of pulmonary fibrosis. The aim of this study was to investigate extracellular superoxide dismutase (ECSOD), the major antioxidant enzyme of the extracellular matrix of human lung, in biopsy-proven idiopathic pulmonary fibrosis (IPF) related to usual interstitial pneumonia (UIP). METHODS AND RESULTS: Fibrotic areas and fibroblastic foci in UIP lungs were notable for absence of ECSOD by immunohistochemistry. Western blotting showed significantly lowered immunoreactivity of ECSOD in fibrotic compared with non-fibrotic areas of the diseased lung. The only cell type that showed intense ECSOD positivity in UIP was the interstitial mast cell. In order to investigate the mechanism for ECSOD depletion in fibrotic areas, alveolar epithelial cells were exposed to tumour necrosis factor-alpha and transforming growth factor (TGF)-beta1; TGF-beta suggested a trend towards decreased synthesis. Patients with UIP were also assessed to determine whether this disease is associated with a naturally occurring mutation in ECSOD (Arg213Gly) which leads to a loss of tissue binding of ECSOD. No significant differences could be found in the allele or genotype frequencies of this polymorphism between 63 UIP patients and 61 control subjects. CONCLUSION: Overall, consistent with several other antioxidant enzymes, ECSOD is very low in fibrotic areas of UIP, which may further increase the oxidant burden in this disease.

Solute traffic across mammalian peroxisomal membrane--single channel conductance monitoring reveals pore-forming activities in peroxisomes.

Mouse liver peroxisomes were isolated by centrifugation in a self-generated Percoll gradient followed by an Optiprep density gradient centrifugation. Peroxisomes contributed 90-96% of the total protein content in the fraction, as confirmed by marker enzyme assays, protein pattern in SDS-PAGE, immunoblotting, and electron microscopy. Solubilized peroxisomal membrane proteins were reconstituted into a planar lipid bilayer. A single-channel conductance monitoring of the reconstituted lipid bilayer revealed the presence of two pore-forming components with a conductance in 1 M KCl of 1.3 nS and 2.5 nS. Control experiments with fractions enriched in mitochondria, lysosomes, and fragments of endoplasmic reticulum showed that the peroxisomal channel-forming activities were not due to admixture of isolated peroxisomes with other cellular organelles. The peroxisomal channels were well preserved in membrane preparations but became unstable after solubilization from the membranes by detergent.

Activation and relocalization of caspase 3 during the apoptotic cascade of human mesothelioma cells.

Apoptosis plays an important role in cancer biology. We investigated the expression of caspases 3 and 8 in malignant mesothelioma and malignant mesothelioma cell lines and putative changes in their ultrastructural expression prior and after exposure to epirubicin. Further studies were conducted to compare these changes to the localization and expression of the bcl-2 group of proteins bcl-X, bax and mcl-1, and Fas-Fas ligand in the same cells. In the histological samples, caspase 3 and 8 immunoreactivity was seen in 27/37 (73%) and 16/37 (43%) of the mesotheliomas. The immunostaining was cytoplasmic diffuse, granular, and occasionally nuclear. All six mesothelioma cell lines expressed caspases 3 and 8 by immunoblotting. After exposure to epirubicin the extent of apoptosis was increased in all cell lines investigated, being weakest in the most resistant M38K cell line. Immunoelectron microscopy revealed immunogold labeling for caspases 3 and 8 in the mitochondria with the accumulation of caspase 3 in the apoptotic bodies, while the mitochondrial localization of the bcl-2 proteins appeared to be very stable. Fas receptor could be detected by flow cytometry, whereas the most resistant cell line (M38K) lacked Fas ligand when assessed by RT-PCR. These results suggest the importance of caspase 3 during the apoptotic process of mesothelioma cells and indicate that epirubicin-induced apoptosis is independent of the mitochondrial pathway.

Overexpression of peroxiredoxins I, II, III, V, and VI in malignant mesothelioma.

Peroxiredoxins (Prxs) are a recently characterized group of thiol-containing proteins with efficient antioxidant capacity, capable of consuming hydrogen peroxide in living cells. Altogether six distinct Prxs have been characterized in mammalian tissues. Their expression was investigated in histological samples of mesothelioma and in cell lines established from the tumours of mesothelioma patients. Four cases with histopathologically healthy pleura from non-smokers were used as controls. Healthy pleural mesothelium was negative or very weakly positive for all Prxs. In mesothelioma, the most prominent reactivity was observed with Prxs I, II, V, and VI. Prx I was highly or moderately expressed in 25/36 cases, the corresponding figures for Prxs II-VI being 27/36 (Prx II), 13/36 (Prx III), 2/36 (Prx IV), 24/36 (Prx V), and 30/36 (Prx VI). Positive staining was observed both in the cytosolic and the nuclear compartment, with the exception of Prx III, which showed no nuclear reactivity. The staining pattern of Prxs III and V was granular. Immunoelectron microscopic localization of Prxs was in accordance with the immunohistochemical findings, showing diffuse cytoplasmic localization for Prxs I, II, IV, and VI and distinct mitochondrial labelling for Prxs III and V. There was no significant association between the extent of staining and different Prxs. It appeared that Prxs may not have prognostic significance, but being prominently expressed in most mesotheliomas these proteins, at least in theory, may play a role in the primary drug resistance of this disease.

Effects of src kinase and TGFbeta1 on the differentiation and morphogenesis of MDCK cells grown in three-dimensional collagen and Matrigel environments.

This study attempted to analyse in detail the effect of src kinase on the growth and differentiation of MDCK cells in different extracellular matrix (ECM) environments. A method was developed to label the membrane proteins in situ and the distribution of cytoskeletal and junctional proteins was visualized in three-dimensional cell complexes, using optical sections generated by confocal microscopy. Independently of the ECM, non-transformed MDCK cells formed differentiated cell cysts with one or a few lumina, with the apical side facing the lumen; ZO-1 was expressed at the tight junctions close to the apical side and beta-catenin, E-cadherin and fodrin along the entire lateral walls. The phenotype of src kinase activated MDCK cells was strongly dependent on the ECM and varied from an irregular cluster in collagen I, to tubular structures in laminin or proteoglycans, and finally to a polarized cell cyst in Matrigel. In collagen I, E-cadherin and beta-catenin were seen partially along the lateral walls and partially in the cytoplasm of src-transformed MDCK cells; fodrin was released into the cytoplasm and ZO-1 was not visualized. When the src-transformed cells were cultivated in Matrigel, their junctional proteins were recruited to the cell membranes and ZO-1 reappeared at the apical face. Thus, the components of Matrigel could overcome the deleterious effect of src on the polarity of MDCK cells. TGFbeta1, together with its receptors and other soluble factors in Matrigel, were responsible for the induction of differentiation. The results show that tyrosine phosphorylation sensitizes the epithelial MDCK cells to ECM and TGFbeta1.

Lack of cytosolic and transmembrane domains of type XIII collagen results in progressive myopathy.

Type XIII collagen is a type II transmembrane protein found at many sites of cell adhesion in tissues. Homologous recombination was used to generate a transgenic mouse line (Col13a1(N/N)) that expresses N-terminally altered type XIII collagen molecules lacking the short cytosolic and transmembrane domains but retaining the large collagenous ectodomain. The mutant molecules were correctly transported to focal adhesions in cultured fibroblasts derived from the Col13a1(N/N) mice, but the cells showed decreased adhesion when plated on type IV collagen. These mice were viable and fertile, and in immunofluorescence stainings the mutant protein was located in adhesive tissue structures in the same manner as normal alpha1(XIII) chains. In immunoelectron microscopy of wild-type mice type XIII collagen was detected at the plasma membrane of skeletal muscle cells whereas in the mutant mice the protein was located in the adjacent extracellular matrix. Affected skeletal muscles showed abnormal myofibers with a fuzzy plasma membrane-basement membrane interphase along the muscle fiber and at the myotendinous junctions, disorganized myofilaments, and streaming of z-disks. The findings were progressive and the phenotype was aggravated by exercise. Thus type XIII collagen seems to participate in the linkage between muscle fiber and basement membrane, a function impaired by lack of the cytosolic and transmembrane domains.

Abnormal adherence junctions in the heart and reduced angiogenesis in transgenic mice overexpressing mutant type XIII collagen.

Type XIII collagen is a type II transmembrane protein found at sites of cell adhesion. Transgenic mouse lines were generated by microinjection of a DNA construct directing the synthesis of truncated alpha1(XIII) chains. Shortened alpha 1(XIII) chains were synthesized by fibroblasts from mutant mice, and the lack of intracellular accumulation in immunofluorescent staining of tissues suggested that the mutant molecules were expressed on the cell surface. Transgene expression led to fetal lethality in offspring from heterozygous mating with two distinct phenotypes. The early phenotype fetuses were aborted by day 10.5 of development due to a lack of fusion of the chorionic and allantoic membranes. The late phenotype fetuses were aborted by day 13.5 of development and displayed a weak heartbeat, defects of the adherence junctions in the heart with detachment of myofilaments and abnormal staining for the adherence junction component cadherin. Decreased microvessel formation was observed in certain regions of the fetus and the placenta. These results indicate that type XIII collagen has an important role in certain adhesive interactions that are necessary for normal development.

Prolyl 4-hydroxylase isoenzymes I and II have different expression patterns in several human tissues.

Prolyl 4-hydroxylase plays a central role in the synthesis of all collagens. We have previously reported that the recently identified Type II isoenzyme is its main form in chondrocytes and possibly in capillary endothelial cells, while Type I is the main form in many other cell types. We report here that the Type II isoenzyme is clearly the main form in capillary endothelial cells and also in cultured umbilical vein endothelial cells, whereas no Type I isoenzyme could be detected in these cells by immunostaining or Western blotting. The Type II isoenzyme was also the main form in cells of the developing glomeruli in the fetal kidney and tubular structures of collecting duct caliber in both fetal and adult kidney, in occasional sinusoidal structures and epithelia of the bile ducts in the liver, and in some cells of the decidual membrane that probably represented invasive cytotrophoblasts in the placenta. Osteoblasts in a fetal calvaria, i.e., a bone developing by intramembranous ossification, stained strongly for both types of isoenzyme. The Type I isoenzyme was the main form in undifferentiated interstitial mesenchymal cells of the developing kidney, for example, and in fibroblasts and fibroblastic cells in many tissues. Skeletal myocytes and smooth muscle cells appeared to have the Type I isoenzyme as their only prolyl 4-hydroxylase form. Hepatocytes expressed small amounts of the Type I enzyme and very little if any Type II, the Type I expression being increased in malignant hepatocytes and cultured hepatoblastoma cells. The data suggest that the Type I isoenzyme is expressed especially by cells of mesenchymal origin and in developing and malignant tissues, whereas the Type II isoenzyme is expressed, in addition to chondrocytes and osteoblasts, by more differentiated cells, such as endothelial cells and cells of epithelial structures. (J Histochem Cytochem 49:1143-1153, 2001)

Surfactant proteins A and D in Eustachian tube epithelium.

Surfactant protein (SP) A and SP-D are collectins that have roles in host defense. The Eustachian tube (ET) maintains the patency between the upper airways and the middle ear. Dysfunction of local mucosal immunity in ET may predispose infants to recurrent otitis media. We recently described preliminary evidence of the expression of SP-A and SP-D in the ET. Our present aim was to establish the sites of SP-A and SP-D expression within the epithelium of the ET in vivo. With in situ hybridization, electron microscopy, and immunoelectron microscopy, the cells responsible for SP-A and SP-D expression and storage were identified. SP-A expression was localized within the ET epithelium, and the protein was found in the electron-dense granules of microvillar epithelial cells. Being concentrated in the epithelial lining, only a few cells revealed intracellular SP-D, and it was not associated with granules. The SP-A and SP-D immunoreactivities in ET lavage fluid, as shown by Western blot analyses, were similar to those in bronchoalveolar lavage fluid. We propose that there are specialized cells in the ET epithelium expressing and secreting SP-A and SP-D. SP-A and SP-D may be important for antibody-independent protection of the middle ear against infections.

Biologic markers in ductal carcinoma in situ and concurrent infiltrating carcinoma. A comparison of eight contemporary grading systems.

The relevance of 8 contemporary classification and grading systems for ductal carcinoma in situ (DCIS) of the breast was examined in 100 tumors by comparing DCIS grade with grade of the concurrent infiltrating ductal carcinoma (IDC). Besides tumor size and nodal status, the immunohistochemical parameters in both lesions were compared, including estrogen receptor, progesterone receptor, c-erbB-2 protein, E-cadherin, vimentin, Ki-67 (MIB1), and p27. Nuclear grading of DCIS alone or in combination with architectural pattern and necrosis showed the best correlation with grade of the invasive component. There also was a positive correlation between every biologic marker expressed in DCIS and in the concurrent IDC, supporting a clonal relationship. Biologic markers varied between the different grades of DCIS. DCIS is heterogeneous, and the progression of DCIS to IDC may be from low-grade DCIS to low-grade IDC and high-grade DCIS to high-grade IDC. This concept is different from the conventional model held for intraepithelial neoplasia in the cervix, vulva, vagina, and skin, in which there is increasing severity of in situ atypia (dysplasia) before the development of stromal invasion.

Expression and prognostic significance of catalase in malignant mesothelioma.

BACKGROUND: Free radicals and antioxidant enzymes (AOEs) may play a critical role in cell proliferation and in the resistance of malignant cells against cytotoxic drugs and radiation. Malignant mesothelioma is a resistant tumor with high levels of manganese superoxide dismutase, a central superoxide scavenging AOE. In the current study, the authors assessed the expression and prognostic role of catalase, an important hydrogen peroxide scavenging AOE, in malignant pleural mesothelioma. METHODS: Catalase expression was investigated by immunohistochemistry in 5 cases of nonmalignant healthy pleura and in tumor tissue of 32 mesothelioma patients, and by Western blot in 7 continuous human mesothelioma cell lines. The distribution of catalase in mesothelioma cells was assessed by immunoelectron microscopy. Furthermore, to investigate the effect of catalase inhibition in the drug resistance of these cells in vitro, the authors exposed mesothelioma cells with the highest catalase level to epirubicin with and without aminotriazole pretreatment. RESULTS: Nonmalignant mesothelial cells showed no catalase immunoreactivity whereas most mesothelioma cases (24 of 32, 75%) were catalase positive, 17 cases (53%) showing moderate or high expression. Higher catalase expression in mesothelioma was associated with a better prognosis, mean survival rate from diagnosis being 6 and 24 months for negative/low expression and moderate/high expression, respectively. Furthermore, a coordinately high expression of both manganese-superoxide dismutase (Mn-SOD) and catalase predicted even more favorable outcome of the mesothelioma patients. Catalase also could be detected in all mesothelioma cell lines, the most resistant cell line showing the highest protein expression and compartmentalization of catalase mainly to peroxisomes. Aminotriazole inhibition of catalase had a marginal effect on the toxicity caused by epirubicin. CONCLUSIONS: Catalase may have multifactorial effects in malignant cells; high catalase and/or coordinated high expression of Mn-SOD and catalase may decrease tumor progression by modulating the cellular redox state, but enhanced antioxidant capacity of mesothelioma cells also may protect tumor cells against exogenous oxidants, at least in vitro.

Expression and localization of lung surfactant protein B in Eustachian tube epithelium.

Surfactant protein (SP) B is an essential component of the pulmonary surfactant complex, which participates in reducing the surface tension across the alveolar air-liquid interface. The Eustachian tube (ET) connects the upper respiratory tract to the middle ear, serving as an intermittent airway between the pharynx and the middle ear. Recently, we described the expression of SP-A and SP-D in the ET, suggesting their role in middle ear host defense. Our present aim was to detect whether the expression of SP-B is evident in the porcine ET. With Northern blot analysis, RT-PCR, and in situ hybridizations, SP-B mRNA was identified and localized in the ET epithelium. The cellular localization of SP-B was revealed with immunohistochemistry, electron microscopy, and immunoelectron microscopy. The protein was found in the secretory granules of epithelial cells and also attached to the microvilli at the luminal side of these cells. The SP-B immunoreactivity of aggregates isolated from ET lavage fluid was similar to that isolated from bronchoalveolar lavage fluid. We conclude that there are specialized cells in the ET epithelium expressing and secreting SP-B and propose that SP-B may facilitate normal opening of the tube and mucociliary transport.

Lack of type XV collagen causes a skeletal myopathy and cardiovascular defects in mice.

Type XV collagen occurs widely in the basement membrane zones of tissues, but its function is unknown. To understand the biological role of this protein, a null mutation in the Col15a1 gene was introduced into the germ line of mice. Despite the complete lack of type XV collagen, the mutant mice developed and reproduced normally, and they were indistinguishable from their wild-type littermates. However, Col15a1-deficient mice showed progressive histological changes characteristic for muscular diseases after 3 months of age, and they were more vulnerable than controls to exercise-induced muscle injury. Despite the antiangiogenic role of type XV collagen-derived endostatin, the development of the vasculature appeared normal in the null mice. Nevertheless, ultrastructural analyses revealed collapsed capillaries and endothelial cell degeneration in the heart and skeletal muscle. Furthermore, perfused hearts showed a diminished inotropic response, and exercise resulted in cardiac injury, changes that mimic early or mild heart disease. Thus, type XV collagen appears to function as a structural component needed to stabilize skeletal muscle cells and microvessels.

Localization of salmon cardiac peptide (sCP) in the heart of salmon (Salmo salar L.).

We have previously cloned and characterized a novel cardiac hormone from the salmon (Salmo salar) which has a uniquely heart-specific distribution and a low structural similarity with any other known natriuretic peptides. Specific antibodies were raised in goat against the salmon cardiac peptide. For localization and quantification, four different methods were applied: immunohistochemistry (avidin-biotin peroxidase), transmission electron microscopy, cryoimmunoelectron microscopy (protein A-gold), and a specific radioimmunoassay. Both atrial and ventricular myocytes stained immunohistochemically. The staining was similar in all myocytes and no specific myoendocrine cells were found. Within a single myocyte, both atrial and ventricular, the staining was stronger near the nucleus. Transmission electron microscopy revealed that both the atrium and the ventricle contained small sarcoplasmic granules of similar type with a diameter of 100 to 200 nm and an electron-dense core with a clear halo. The granules were typical vesicles which can be found in secretory cells utilizing the regulatory pathway. The highest number of granules was found near the nucleus, but granules were located also near the Golgi apparatus, between myofilament bundles, and in subsarcolemmal positions. Gold particles, conjugated to antibodies raised against the salmon cardiac peptide, were deposited on similar sarcoplasmic granules found in transmission electron microscopy. Among the sarcoplasmic granules with gold particles there were granules which did not show any cardiac peptide immunoreactivity. A significantly (Student's t test, P < 0.05) higher concentration of cardiac peptide was found in the heart atrium than in the ventricle, 16.2 +/- 3.5 pmol/mg tissue (n = 8) and 4.5 +/- 1.7 pmol/mg tissue (n = 8), respectively. The findings show that the salmon cardiac peptide is localized in secretory granules in both compartments of the heart. The morphology of the granules suggests that both the atrium and the ventricle utilize the regulatory pathway to release salmon cardiac peptide.

Immunolocalization of the fodrin, E-cadherin, and beta-catenin adhesion complex in infiltrating ductal carcinoma of the breast-comparison with an in vitro model.

Fodrin, E-cadherin, and beta-catenin immunolocalization was studied in 54 cases of infiltrating ductal carcinoma of the breast and compared with an in vitro model in order to study the dynamic relationship between these components of an adhesion complex. In low-grade tumours, the staining patterns were similar for both fodrin and E-cadherin, with localization of these proteins to the cell membranes. beta-Catenin showed reduced membrane staining compared with non-neoplastic epithelium. High-grade tumours displayed strong membranous as well as cytoplasmic immunolocalization of fodrin, while E-cadherin staining was fragmented or lost from the membranes, with only occasional weak intracellular staining. beta-Catenin showed fragmented membrane staining and cytoplasmic accumulation. In addition, nuclear staining of beta-catenin was occasionally observed. In a v-src-transformed MDCK cell line, following 15min of src activation, beta-catenin began to detach from the cell membrane and localize to the cytoplasm, while fodrin and E-cadherin remained unchanged. After 30-45min of src activation, the cells lost their cuboidal shape and began to lose cell-to-cell contact. Fodrin staining remained mostly membranous while that of E-cadherin and beta-catenin was fragmented and spiky. After 60min of src activation, fodrin localized completely in the cell cytoplasm, while E-cadherin and beta-catenin were partly cytoplasmic with fragmented and spiky membranous staining. Occasionally, beta-catenin was seen in the nucleus. Both in vivo and in vitro findings clearly demonstrated a disruption of the E-cadherin/beta-catenin/fodrin/cytoskeleton linkage concomitant with the loss of cell-to-cell adhesion and change in cell shape, from epithelioid to a fibroblastoid phenotype. Membranous localization of E-cadherin showed a positive correlation with oestrogen and progesterone expression, whereas loss of membranous E-cadherin and cytoplasmic accumulation of fodrin was more often observed in high-grade carcinomas and showed a positive correlation with p53 expression.