Effect of collagenase on surface expression of immunoreactive fibronectin and laminin in freshly isolated cardiac myocytes.

The extracellular glycoproteins fibronectin (FN) and laminin (LMN) are ubiquitously expressed in myocardial tissue. These glycoproteins are important for cellular attachment and differentiation of the cardiac myocytes. Utilizing specific antibodies for the detection of FN and LMN, respectively, the distribution of these extracellular proteins was examined in enzymatically isolated adult cardiac myocytes. Immunofluorescence staining of rod-shaped cardiac myocytes revealed only remnants of immunoreactive FN on the cellular surface and in the transverse tubular membrane system. LMN expression, however, was preserved in a raster-like pattern in the cardiac myocytes. In order to study the distribution of these glycoproteins at high resolution, scanning electron microscopy using the backscattered electron mode was combined with immunogold staining and silver-enhancement. In addition, to confirm the immunofluorescence microscopic observations it was shown that FN labelling was restricted to ill-defined extracellular material and that LMN was absent from the intercalated discs of the cardiac myocytes. The hypercontracted cells were characterized by numerous surface protrusions devoid of immunoreactive LMN. Thus, these results indicate that FN and LMN are differently affected by collagenase treatment, and that these changes of glycoprotein expression may influence the normal function of the cardiac myocytes as well as the membrane stability during the development of irreversible cellular lesions.

Mast cell and mast cell granule phenotypes in normal and Nippostrongylus-infected rats. A qualitative laser confocal microscopic study.

In the rat, the individual mast cell secretory granules may be divided into three subpopulations based on the presence of the specific proteases RMCP-1, RMCP-2, or a variable combination of these two proteases. Mast cells in the tongue only express RMCP-1, both in normal and infected animals, whereas in the other tissue locations studied (lung, intestinal mucosa and submucosa, tracheal epithelium and submucosa) the mast cells contain all three granule subtypes in a wide variation of combinations. These studies demonstrate that there is wide heterogeneity in protease expression in rat mast cells, which may be influenced by local stimulation with environmental tissue factors.

Expression of fibronectin, laminin and ribosomes in normal and nocodazole-treated neonatal heart cells in culture: a study by laser scanning confocal microscopy and immunocytochemistry.

Polyclonal and monoclonal antibodies were used to examine the effects of the synthetic microtubule disruptive drug nocodazole on the subcellular expression of fibronectin, laminin, and ribosomes in primary cultures of neonatal cardiac ventricular cells. Non-invasive serial optical sectioning was carried out by immunolaser scanning confocal microscopy. In addition, fibronectin and laminin were immunolabelled with peroxidase or gold conjugates for electron-microscopic examination. Immunolabelling for the large 60S ribosome subunit in fibroblast-like non-myocytes showed that punctate ribosome structures with a multi-subunit composition were present in perinuclear region. Double immunostaining with antibodies directed against ribosomes and cellular fibronectin indicated that the punctate structures were cisternae of the rough endoplasmic reticulum. No clear effects of nocodazole treatment were detected on the distribution of cytoskeleton-bound ribosomes. Following immunolabelling for both glycoproteins and double immunolabelling for cellular fibronectin and the 60S ribosome subunit, fibronectin and laminin were found in the perinuclear cisternae of the rough endoplasmic reticulum and in pleomorphic secretory vesicles. The cisternal stacks of the Golgi complex appeared either unstained or were only weakly labelled. When these cells were exposed to nocodazole, fibronectin and laminin accumulated in peripheral parts of the cytoplasm, including cellular processes. These peripheral accumulation of immunostaining for fibronectin and laminin did not reflect Golgi staining, as shown by double labelling experiments versus wheat-germ-agglutinin staining, and, by exposing cultures to a high dose of brefeldin A.

Induction of colonization factor antigen I (CFA/I) and coli surface antigen 4 (CS4) of enterotoxigenic Escherichia coli: relevance for vaccine production.

Regulatory proteins control the expression of the fimbrial colonization factor antigens CFA/I and CS4 of enterotoxigenic Escherichia coli (ETEC). To examine the mechanism behind lack of expression of these antigens in spontaneous CFA-negative mutants, we mobilized a recombinant plasmid harbouring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 expression, into such derivatives. In electron microscopy, the induced surface structures were morphologically identical to the fimbriae of the CFA/I+ and CS4+ wild type strains. Immunogold labelling with monoclonal antibodies showed that the distribution of CFA/I and CS4 specific epitopes along the induced fimbriae was indistinguishable from that of the wild type strains. The percentage of fimbriated cells was consistently higher in the cfaD transformants than in the corresponding wild type strains. The present work reports on the efficiency of the cloned cfaD gene in restoring and enhancing the production of morphologically intact CFA/I and CS4 fimbriae.

Immunofluorescence and immunogold electron microscopy of desmin in isolated adult heart myocytes of the rat.

Isolated myocytes of the adult mammalian heart are useful for studying cytoskeletal changes during development of irreversible myocardial injuries. Using monoclonal antibodies we have studied the structural organization of desmin in freshly isolated cardiomyocytes from rat hearts. This preparation consists of approximately 85% calcium tolerant rod shaped cells and 15% contracted "square cells" and "round cells" that were initially injured during separation. Cells were quick-frozen at -196 degrees C without any chemical stabilization, cryosectioned and then further processed for immunofluorescence or immunoelectron microscopy. Freshly isolated rod shaped cells exhibit the specific pattern of interfibrillar desmin organization of striated muscle. Furthermore, high resolution immunogold preparations show that desmin in the rod cells occurs in apposition to the edges of the Z-bands as well as closely associated with the plasmalemma. We could find no evidence for the presence of desmin within the Z-band plaques. This organization of desmin is completely absent in the contracted round cells. Thus, already at advanced stages of square cell development, desmin is almost entirely confined to the outer areas of the central filamentous core. We conclude that during the process of square cell contracture, the filamentous desmin contacts with Z-bands and sarcolemma are broken, leading to the unorganized array of desmin in round cells.

Myocardial cAMP and calcium levels in the calcium paradox.

In a graded model (minimal, subtotal, total) of the calcium paradox phenomenon induced by a progressive increase in the flow rate and volume (5 ml, 10 ml, 45 ml) of calcium-free perfusion (5 min) the severity of tissue injury on calcium repletion was related to a potential elevation of cAMP during calcium depletion. In the subtotal and total models of the calcium paradox a 50% increase was found for tissue cAMP after calcium-free perfusion, but no such rise could be associated with the minimal calcium paradox. In the study tissue injury, as assessed by whole tissue and mitochondrial accumulation of calcium and by myocardial leakage of creatine kinase, could only partly be related to cAMP changes. It is concluded that calcium-free coronary perfusion induces a complex series of events favouring excessive calcium entry, only one of which may be related to a change in cAMP.

Influence of plasma proteins on erythrocyte morphology and sedimentation.

The influence of plasma proteins on erythrocytes was studied by interference microscopy, scanning electron microscopy (SEM), and by Westergren erythrocyte sedimentation rate (ESR). Albumin kept erythrocytes dispersed as discoid spheres. Fibrinogen seemed responsible for the rouleaux phenomenon, but needed the co-influence of an immunoglobulin to induce rouleaux type of aggregates and high ESR. IgG, IgA and IgM caused immunologic type of aggregates. Albumin acted synergistically with fibrinogen and immunoglobulins. Normal blood contained a network of rouleaux, which probably explained the low normal ESR. High ESR was either due to rouleaux type aggregates where fibrinogen was dominant, or immunologic type aggregates where IgG, IgA or IgM were dominant proteins. Cold agglutinin disease showed normal blood morphology and normal ESR at 37 degrees C and immunologic type aggregates and high ESR at 25 degrees C.

A procedure for selecting specific areas of sections for comparative light and electron microscopic studies.

The paper describes a method which enables correlative light and electron microscopical studies to be made of specific tissue structures or of individual cells. For this purpose a new specimen marker has been constructed. The device, which is attached to the objective of a light microscope, permits the operator to examine and exactly encircle the selected area without changing the objective. The technique is illustrated by two examples using immunoelectronmicroscopy.

Protective effects of verapamil against isoprenaline- induced mobilization of mitochondrial calcium and cellular lipid droplets in the myocardium.

Calcium level and lipid droplets accumulation is studied in the mouse myocardium of animals receiving a single injection of isoprenaline (ISP), of animals receiving ISP after 4-8 days of pretreatment with verapamil (Isoptin), and, of control animals. The animals are sacrificed 4h after the ISP-injection. Calcium is determined in isolated mitochondria and in whole tissue homogenates by atomic absorption spectrometry. Lipid droplets accumulation is analysed by quantitative stereological technique. Mitochondrial calcium is raised by 65% and calcium of whole tissue homogenates by 21% following ISP stimulation. At the same time, the myocardial fractional volume occupied by lipid droplets in the ISP treated animals increases to 14 times that of control. The ISP stimulated elevation of mitochondrial calcium is reduced by 73% and that of lipid fractional volume by 71% after pretreatment with verapamil. No similar reduction of calcium content is observed in the whole tissue homogenates. These results are discussed in terms of the role of Ca2+ in ISP induced myocardial necrosis.

Calcium and magnesium levels in isolated cardiac mitochondria from mice injected with isoproterenol.

Calcium (Ca) and Magnesium (Mg) are determined by atomic absorption flame spectrometry in isolated cardiac mitochondria from mice receiving subcutaneous injections of DL-isoproterenol HCl (ISO), and in mitochondria of untreated controls. In the controls, mitochondria were isolated in the presence or absence of ruthenium red. On the absence of ruthenium red in the isolation medium, mitochondrial Ca levels increase by about 300%, while levels of Mg remain unchanged. Focal myocardial necrosis following a single ISO-injection is shown by electron microscopy. Ca and Mg levels are largely unaffected by a single dose of ISO until 24 h after the injection. A slight increase in Ca occurs in the 48 h samples. When multiple injections of ISO are given every 12th hour of 48 h, 72 h and 96 h, respectively, endogenous Ca and Mg increase significantly. It is suggested that this increase might be associated with ISO-induced cardiac hypertrophy rather than with the pharmacological effects of ISO per se.

Patterns in quench-frozen, freeze-dried, blood proteins.

Scanning electron micrographs of whole blood prepared with cryotechnics demonstrated patterns assumed to be artifacts of the freezing process. A SEM investigation of the effects of freezing and freeze-drying on columns of purified blood proteins has been carried out using quench-freezing and cryo-fracturing followed by freeze-drying. Correlative studies with a light microscope and a transmission electron microscope are included. The resulting patterns depended on the type and concentration of protein as well as on the position within the sample and the orientation of the fracture plane relative to the column axis.

Calcium and magnesium levels in isolated mitochondria from human cardiac biopsies.

A non-enzymatic method is presented for isolating mitochondria from small-sized human cardiac samples, including ventricular needle biopsies of 15-25 mg of wet weight. Electron microscopy demonstrates that these fractions are rich in structurally well preserved mitochondria. Calcium and magnesium levels of fractions are determined by atomic absorption flame spectroscopy. Comparative analyses are made in similar fractions of the mouse ventricle. Calcium concentrations of mitocondria isolated in the presence of ruthenium red do not differ significantly between the human auricle and ventricle, averaging 61 nmol Ca/mg protein and 68 nmol Ca/mg protein, respectively. Mitochondrial calcium level is lower in the mouse ventricular fractions, averaging 7 nmol Ca/mg protein. Mitochondrial magnesium amounts to slightly less than 60% of the calcium levels in the human heart, while it exceeds the calcium level by more than 100 per cent in the mouse heart. There is no significant difference of mitochondrial calcium between normal auricles, and, auricles of patients with increased right atrial mean pressure and/or volume overload.

Preservation of shock-frozen myocardial tissue as shown by cryo-ultramicrotomy and freeze-fracture studies.

Myocardial tissue was quickly quench-frozen by letting the specimen fall either into liquid nitrogen (LN2) or into LN2-slush, or, by drill-propelling the specimen into LN2 at a speed of 1.5 m/s. The preservation of the tissues was studied in ultrathin, dry-cut and freeze-dried cryo-sections. Shock-freezing by propelling into LN2 yields extensive areas of well preserved tissue without hole damage. Quenching into LN2-slush shows variable results, while a maximum of hole damage is obtained by letting the specimen fall into LN2. Replicas of freeze-fractured tissue which had been quench-frozen by the same methods confirmed these observations. By the drill-propelling, areas 2000-5000 micrometer2 wide reveal good preservation without visible ice crystals. Such areas occur within a superficial band of tissue to a depth of c. 20 micrometer. Even at 35 micrometer and 50 micrometer depth good preservation may be registered at the periphery of the replicas. Description is given of modifications in accessories and methods in order to make the LKB CryoKit more suitable as a routine instrument. These modifications cover replacement of the knife coolant container by a coolant brass container in open connection with three copper tubes, replacement of one of the LKB Dewar flasks by a 25 litre Dewar equipped with a Balzers filling device, and replacement of the LKB plastic sleeve fixed to the back of the specimen coolant container, by two perspex-glass discs mounted in the slit between the Ultrotome base and the cryo-chamber. In addition, modified constructions are presented of the grid-carrier, the press-assembly as well as of the container for freeze-drying and warming up of the frozen sections.