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N J Severs

Publications and source records attributed to N J Severs.

At least 127 records · Page 7Linked to original sources
PubMed1985

Morphometric analysis of calcium-tolerant myocytes isolated from the adult rat heart.

The ultrastructure of calcium-tolerant isolated myocytes has been compared with that of intact heart myocytes using morphometric techniques. Isolated myocytes were prepared by the method of Powell et al. [1] from four adult rat hearts, and multiple left ventricular tissue samples were obtained from a further four rat hearts after fixation by Langendorff perfusion. The subcellular-component volumes of myofibrils, mitochondria, nuclei, transverse tubules, sarcoplasmic reticulum, lipid droplets, and cytoplasmic space have been estimated using a point-counting method on thin sections of intact myocardium and isolated myocytes. No significant differences between the two sample types were found. With the use of freeze-fracture, the numerical density and distribution of sarcolemmal intramembrane particles were analyzed by the method of Jones et al. [2]. The intramembrane particle density of isolated myocyte E-faces was very similar to that of myocytes of intact myocardium. However, isolated myocytes showed a slight reduction in P-face intramembrane particle density (less than 10%), although intramembrane particle distribution remained unaltered on both fracture faces. Our findings show that the ultrastructural features of the isolated myocytes closely resemble those of their counterparts in the intact myocardium.

Animals↗
PubMed1985

Ultrastructure of the sarcolemma and intercalated disc in isolated rat myocytes.

The ultrastructure of the sarcolemma in isolated calcium-tolerant myocytes has been compared with that of myocytes in the intact heart, using thin section and freeze-fracture electron microscopical techniques. Data on the density and distribution of intramembrane particles and on the topography of Z-folds in the general (i.e. non-disc) sarcolemma are summarised. The fate of gap junctions on separation of the intercalated disc membranes has been studied i) at intervals after isolation, ii) using cationized ferritin as an extracellular marker and iii) by serial sectioning. The results of these studies help explain how ionic integrity of the individual isolated cells may be maintained.

Animals↗
PubMed1985

Rough endoplasmic reticulum in the adult mammalian cardiac muscle cell.

The morphology, distribution and extent of rough endoplasmic reticulum (RER) was investigated in normal and ischaemic left ventricular 'working' myocytes of the adult rabbit. Continuity of RER with membranes of the Golgi apparatus, nuclear envelope and sarcoplasmic reticulum (SR) is documented, though 48% of RER profiles appear as discrete entities in the interfibrillar and perinuclear sarcoplasm. Of the RER intercalated in the SR, 31% of the segments occur adjacent to the Z-tubule, 15% in longitudinal elements of free SR and 5% adjacent to junctional SR. Quantitative analysis of profile lengths shows the RER to be more extensive than hitherto suspected, with 73% of profiles having lengths in excess of 200 nm and some (albeit uncommonly) in excess of 8 micron.

Animals↗
PubMed1984

Connexon rearrangement in cardiac gap junctions: evidence for cytoskeletal control?

Using ultrarapid-freezing techniques and freeze-fracture electron microscopy, we report here a close association between cardiac gap junctions and specialized membrane domains containing regularly-spaced furrows. These specialized furrowed domains are observed only during periods of gap junction re-organisation (i.e., connexon redistribution) and may reflect the presence of underlying cytoskeletal elements controlling the position of connexons in the membrane.

Animals↗
PubMed1984

Double labeling of lipoprotein receptors in fibroblast cell surface replicas.

Using gold particles of distinctly different sizes as markers, the simultaneous visualization of two types of lipoprotein binding sites in surface replicas of the human fibroblast plasma membrane was achieved. Double-labeling experiments based on this principle have been carried out using sequential or simultaneous exposures of fibroblast cultures to a variety of lipoprotein species. Examples of the results obtained are illustrated, and the application of this approach to the analysis of the distribution, the relationship between, and the specificity of different classes of receptor is discussed.

Cell Membrane↗
PubMed1984

The "promoting" activity of methyl methanesulphonate in rat bladder carcinogenesis.

The carcinogenic activity of the alkylating agent methyl methanesulphonate (MMS) was investigated in the F344 rat bladder, both untreated and pretreated with a single threshold dose of N-methyl-N-nitrosourea (MNU). On its own, 6 doses of 2.5 mg MMS produced a 7% incidence of bladder cancer. After a single intravesical instillation of MNU, the same MMS treatment produced a bladder cancer incidence of 56%. This was significantly higher than the incidence (24%) observed after treatment with MNU alone, and greater than the sum of the lesions produced by either treatment alone. By reference to the mouse skin multistage carcinogenesis model, it is argued that MMS is a complete, albeit weak carcinogen with little initiating but powerful late-stage activity. Its promoting activity is most probably attributable to its potent mitogenic action and in this model it is analogous to a stage 2, rather than a stage 1 skin promoter.

Animals↗
PubMed1984

Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks.

By using two ultrarapid freezing techniques, we have captured the structure of rat and rabbit cardiac gap junctions in a condition closer to that existing in vivo than to that previously achieved. Our results, which include those from fully functional hearts frozen in situ in the living animal, show that the junctions characteristically consist of multiple small hexagonal arrays of connexons. In tissue frozen 10 min after animal death, however, unordered arrays are common. Examination of junction structure at intervals up to 40 min after death reveals a variety of configurations including dispersed and close-packed unordered arrays, and hexagonal arrays. By use of an isolated intercalated disk preparation, we show that the configuration of cardiac gap junctions in vitro cannot be altered by factors normally considered to induce functional uncoupling. These experiments demonstrate that, contrary to the conclusions of some earlier studies (Baldwin, K. M., 1979, J. Cell Biol., 82:66-75; Peracchia, C., and L. L. Peracchia, 1980, J. Cell Biol., 87:708-718), the arrangement of gap junction connexons, in cardiac tissue at least, cannot be used as a reliable guide to the functional state of the junctions.

Animals↗
PubMed1983

Freeze-fracture analysis of plasma membranes in Duchenne muscular dystrophy. A study using cultured skin fibroblasts.

Quantitative freeze-fracture studies of the numercial density and distribution of intramembrane particles (IMP) were carried out on the plasma membrane of cultured skin fibroblasts from 4 patients with Duchenne muscular dystrophy and 3 normal controls. Analysis of P and E fracture faces of the fibroblast plasma membrane failed to show any significant differences in either IMP density or distribution between normal and dystrophic specimens. In contrast to previous reports, our results indicate that an altered IMP density is not a characteristic feature of the dystrophic cell membrane.

Cell Adhesion↗
PubMed1983

A simplified method for the rapid isolation of cardiac intercalated discs.

A simple and rapid method for the isolation of intercalated discs from a single rat heart is described. The outstanding features of this method are (i) a large quantity of starting material is not necessary, (ii) ultracentrifugation and separating gradient steps are not required, (iii) the disc fraction is relatively pure as verified by electron microscopy, and (iv) the starting tissue is rapidly homogenized into buffer, reducing possible damage to membrane structures after animal death. The fraction is rich in intercellular junctions, with gap junctions, fasciae adherentes and maculae adherentes all appearing well preserved. It should prove a useful starting base for further purification of these junctions and of sarcolemma from a specific portion of the cell surface membrane, namely the major area of structural interaction between adjacent cardiac cells.

Animals↗
PubMed1983

Matrix constituents of early developing bone examined by freeze fracture.

Specimens of aldehyde-fixed and glycerol-impregnated early developing bone matrix, obtained from rat calvaria, were examined by the freeze-fracture method. The developing bone matrix reveals collagen fibrils, numerous membranous structures and a granular background. The collagen fibrils, when viewed longitudinally, exhibit a substructure of thinner filaments (microfibrils) which appear to follow a twisted course along the fibril-axis. Some of the membranous structures are readily identified as osteoblast processes. Others, which are round, ovoid or irregular in shape, were found either with or without intramembrane particles (IMPs). It is concluded that the round or ovoid IMP-containing membranous structures correspond to matrix-vesicles. The nature of the IMP-free bodies, however, is uncertain. They may be artefacts or genuine matrix-vesicles deriving from unstable membrane domains which have a propensity for blebbing or budding off. Confirmation of the latter possibility might come from examination of directly frozen specimens.

Animals↗
PubMed1983

The induction of urothelial hyperplasia by methyl methanesulphonate and ethyl methanesulphonate.

The early and late morphological changes induced in rat bladder urothelium by intravesicular administration of the alkylating agents methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS) are described. In the short-term, both compounds produced dose-related toxic damage followed by a regenerative hyperplasia of the urothelium. At any given dose-level, the effects of MMS were more severe than those of EMS. Two years after administration of multiple doses of 2.5 mg MMS or 7.5 mg EMS the majority of animals had dose-related simple urothelial hyperplasias with occasional mild dysplasia. However, in three MMS-treated animals the hyperplasias had progressed to well-differentiated transitional-cell carcinomas. No bladder neoplasms were seen in EMS-treated animals. The urothelial response of the rat to MMS and EMS is discussed with reference to the known chemical reactivity of these compounds. It is concluded that EMS is a mitogen for the urothelium and that the few carcinomas which develop following topical exposure of the bladder to MMS do not necessarily reflect any initiating potential in this compound. Rather it is argued that the results are consistent with MMS acting as a promoter in cells which have either been previously initiated or which carry a latent oncogene.

Animals↗
PubMed1983

Isolated calcium-tolerant myocytes and the calcium paradox: an ultrastructural comparison.

The ultrastructure of calcium-tolerant myocytes isolated from adult rat ventricular myocardium is described, using thin section and freeze-fracture electron microscopy. Two distinct cell types are observed, rounded and rod-shaped. The former are damaged myocytes and superficially resemble the disrupted cells characteristic of the calcium paradox. Despite this resemblance however, the genesis of these isolated damaged cells is not explicable in terms of the calcium paradox. The majority of isolated cells are rod-shaped and show well preserved ultrastructural features. Our results indicate that whatever mechanisms underlie the calcium paradox it is not an indispensable condition that isolation of myocytes necessarily leads to this phenomenon.

Animals↗
PubMed1982

Cholesterol distribution and structural differentiation in the sarcoplasmic reticulum of rat cardiac muscle cells. A freeze-fracture cytochemical investigation.

The polyene compound, filipin, was used as a probe to localize cholesterol in the membranes of the rat cardiac muscle cell, with particular reference to the sarcoplasmic reticulum (SR). Filipin binds specifically to cholesterol (and related 3-beta-hydroxysterols) in membranes, producing distinct deformations which can be viewed by freeze-fracture and used as markers for the presence of cholesterol-rich regions in the membrane plane. In freeze-fracture replicas of filipin-treated rat myocardium, the muscle cells revealed abundant deformations in their plasma membranes, no deformations in mitochondrial membranes, and an intermediate response in the SR. These results are in agreement with the levels of cholesterol reported in isolated fractions of the different membrane types, and confirm the specificity of filipin action. Within the SR, the filipin-induced deformations were not randomly distributed but occurred more commonly in free SR at or near the Z-region of the sarcomere than in other parts of the free SR or the junctional SR. This finding is interpreted as evidence for a non-homogeneous distribution of cholesterol in cardiac muscle cell SR. The possible significance of cholesterol in relation to structural differentiation and function of the SR is discussed.

Animals↗
PubMed1982

Induction of bladder cancer in rats by fractionated intravesicular doses of N-methyl-N-nitrosourea.

Experiments were conducted to determine the dose response of rat bladder urothelium to a range of different single and fractionated intravesicular doses of the carcinogen, N-methyl-N-nitrosourea (MNU). A dose-related response of bladder-tumour incidence to single graded doses of MNU was found, and a threshold does suitable for use of multistage carcinogenesis experiments was derived from these data. For any given total dose of MNU, the tumour incidence was greater if the MNU had been administered in several small fractions than if it had been administered in fewer larger ones. Extending the interval between doses did not reduce the tumour incidence. It is argued that these results support the multistage theory of carcinogenesis. The histopathology and cell-surface alterations which characterize the development of MNU-induced bladder cancer are described and the contribution of hyperplasia and calculi are discussed.

Animals↗
PubMed1982

Comparison of the response of myocardial muscle and capillary endothelial nuclear membranes to the cholesterol probe filipin.

The polyene antibiotic filipin was used as a probe for cholesterol in the nuclear membranes of cardiac muscle and capillary endothelial cells. Samples of perfusion-fixed rabbit and rat myocardium were exposed to filipin in glutaraldehyde fixative, prior to examination by standard freeze-fracture techniques. Quantitative analysis of the responses of the inner and the outer nuclear membranes of the two cell types was carried out by measuring the numerical density of filipin-induced lesions in each. Cardiac muscle cell outer nuclear membranes displayed few or no lesions (less than 0.1 micron-2), and their inner nuclear membranes were completely resistant to the treatment. By contrast, a marked response to filipin was found in the endothelial nuclear envelope, the outer membrane showing an average of 250 lesions micron-2, and the inner membrane 63 lesions micron-2. These results are interpreted as a direct reflection of differences in cholesterol content between the outer and inner nuclear membranes of the two cell types. The significance of the apparent differences in cholesterol content demonstrated between the nuclear envelopes of different cell types is discussed with reference to biochemical data, plasma membrane cholesterol content, nuclear membrane function and the supply of exogenous cholesterol.

Animals↗