Paediatricians' views on smacking children as a form of discipline.

Smacking children as a form of discipline is controversial. This study examined British Paediatricians' views on smacking. We found that their views varied and may be influenced by their personal experiences. This may affect their decision to investigate suspected child abuse and the advice given to parents. More teaching for doctors is required on alternative disciplinary methods.

Integrity of the dissociated adult cardiac myocyte: gap junction tearing and the mechanism of plasma membrane resealing.

Dissociation of adult cardiac myocytes by collagenase perfusion techniques requires separation of the junctional contacts that link the cells physically, electrically and metabolically in the intact heart. Gap junctions, one of three types of intercellular junction present at the cardiac intercalated disc, are not split into their component membranes when myocytes are dissociated; they are ripped from the plasma membrane of one cell, to be retained by its neighbour. Partitioning of junctions in this way might be expected to constitute a serious threat to the ionic integrity of dissociated myocytes, but in practice, high yields of functionally intact cells, suitable for experimental studies, are routinely obtained. To explain this apparent paradox, repair mechanisms, operating to seal the membrane lesions caused by gap junction tearing, have been hypothesized, but evidence for their existence has previously been lacking. Using freeze-fracture electron microscopy, the present study identifies repair sites as smooth membrane domains that are continuous with the neighbouring plasma membrane, thus forming intact seals. That these structures are not chemically-induced artefacts is demonstrated by their presence in myocytes that were frozen directly from the living state. Subsarcolemmal vesicle clusters, detected in thin sections and freeze-fracture replicas, are associated with the smooth sealing domains. These structures may represent either rounded-up fragments of mechanically disrupted membrane or structures concerned with the synthesis of new lipid. From their freeze-fracture morphology, the sealing domains appear to be lipid-rich and protein-poor. Cytochemical studies using Ruthenium Red, cationized ferritin and lectins show in addition that they have a lower content of negatively-charged membrane components than the neighbouring plasma membrane, and that the carbohydrate residues normally associated with plasma membrane glycolipids and glycoproteins are absent.

Purification and characterization of three (1----4)-beta-D-xylan endohydrolases from germinated barley.

Three (1----4)-beta-D-xylan xylanohydrolases (xylan endohydrolases, EC 3.2.1.8) have been purified 1200-2800-fold from extracts of germinated barley (Hordeum vulgare L. cv. Clipper) by a sequence of ammonium sulphate fractionation, Procion-blue-dye chromatography, ion-exchange and gel filtration chromatography. The enzymes are likely to function in the depolymerization of cell wall arabinoxylans during mobilization of the starchy endosperm. They are classified as endohydrolases on the basis of analyses of products released during hydrolysis of a (1----4)-beta-xylan. The three xylan endohydrolases are monomeric proteins of apparent Mr 41,000 and all have isoelectric points of 5.2. The sequences of the 30 NH2-terminal amino acids of the three enzymes are the same, but it is not yet known whether they represent the products of separate genes or originate by differences in post-translational modification of a single gene product.

Fate of gap junctions in isolated adult mammalian cardiomyocytes.

The fate of gap junctions in dissociated adult myocytes, maintained for up to 22 hours in culture medium, was investigated by semiquantitative analysis of thin sections and by freeze-fracture electron microscopy. Gap junctions in the dissociated myocyte are intact bimembranous structures seen either as invaginated surface-located structures or as annular profiles in the cytoplasm. Surface-located junctions are sealed from the exterior by a sheet of nonjunctional membrane originating (together with the "outer" junctional membrane) from the formerly neighboring cell. Serial sectioning was used to establish that at least part of the annular gap junction population in the freshly isolated myocyte represents truly discrete cytoplasmic vesicles; thus, some gap junctions are rapidly endocytosed after myocyte separation. Analysis of the surface-located-to-annular gap junction ratio suggested that no further endocytosis occurred in rabbit and cat myocytes maintained for 22 and 15 hours, respectively. Guinea pig myocytes, by contrast, did appear to continue endocytosis in culture. Analysis of the distance of gap junctional structures from the cell surface suggested that little if any inward migration of gap junction vesicles occurred. Hypoxia had no detectable effect on the internalization or inward movement of gap junctions. The quantity of ultrastructurally detectable gap junction membrane appeared to remain constant over time, as did the incidence of "complex structures" (i.e., annular gap junction profiles with features previously suggested to represent degradation). New gap junction formation was negligible, and a reappraisal of the nature of "complex structures" led to the conclusion that the origin of these structures need not be related to degradation. Taken together, the findings suggest that degradation and disappearance of gap junctional membrane after isolation of the mature myocyte constitute a much slower process than previously believed, and the possibility that the cardiac gap junction protein has a longer half-life than its counterpart in liver remains open.

Stretch-induced centrifugal movement of atrial specific granules--a preparatory step in atrial natriuretic peptide secretion.

Atrial natriuretic peptide (ANP) is a circulating hormone produced by atrial muscle cells which acts upon renal vascular smooth muscle to raise glomerular filtration pressure, thereby increasing salt and water excretion and reducing blood pressure [2, 5, 8]. As in other peptide hormone-secreting cells, ANP is packaged and stored in dense Golgi-derived secretory granules [4-6]. These atrial specific granules [6] occur predominantly in large clusters deep in the myocyte's interior, associated with Golgi cisternae in myofibril-free cytoplasm at the nuclear poles [5, 6]. Smaller clusters and individual granules are distributed beneath the sarcolemma and between myofibrils [6, 10]. Expansion of the blood volume raises the level of circulating ANP [7], a response mediated by mechanical stretch of the atrial wall [1, 3, 9]. The mechanism by which cell stretch induces release of ANP from atrial myocytes is, however, unknown. We show here that one component of this unusual secretory mechanism involves a sudden centrifugal movement of atrial specific granules toward the cell surface.

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.

Different effects of thiopental in severe hypoxia, total ischemia, and low-flow ischemia in rat heart muscle.

The effect of thiopental (100 mg X 1(-1] during total ischemia, low-flow ischemia, and severe hypoxia with maintained flow was investigated in the isolated perfused rat heart. During total ischemia the rate of decline of tissue creatine phosphate and adenosine triphosphate was no different in thiopental-treated and untreated hearts. The development of ultrastructural damage during total ischemia, the release of creatine kinase on reperfusion, and the exacerbation of ultrastructural damage after reperfusion were unaffected by thiopental. When thiopental was added to the perfusate during hypoxia and during low-flow ischemia at a normal pH(7.4), creatine kinase release during reoxygenation and during reperfusion was significantly less (P less than 0.005 and P less than 0.05, respectively) than in the untreated groups. After low-flow ischemia at a low pH (6.5), creatine kinase release was no different in thiopental-treated and untreated hearts. Thus, thiopental afforded protection of the myocardium in hypoxia and low-flow ischemia at pH 7.4 but not in total ischemia and low-flow ischemia at pH 6.5. The data are consistent with the hypothesis that during total ischemia and low-flow ischemia at pH 6.5, acidosis favors the entry of thiopental into the cell, causing inhibition of mitochondrial function and reduction of ATP production. During hypoxic perfusion and low-flow ischemia at pH 7.4, when the decrease in pH is less, the cardiodepressant effect of thiopental may offset any deleterious effect of the drug on intracellular organelles such as mitochondria.

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.

Morphometric analysis of the isolated calcium-tolerant cardiac myocyte. Organelle volumes, sarcomere length, plasma membrane surface folds, and intramembrane particle density and distribution.

Using morphometric analysis of thin sections and freeze-fracture replicas, the ultrastructure of isolated rat myocytes prepared by collagenase digestion (Powell et al. 1980) was compared with that of myocytes fixed by perfusion of intact myocardium. The volumes of myofibrils, mitochondria, nuclei, sarcoplasmic reticulum and lipid droplets in the isolated myocytes did not differ from those of their counterparts in the intact heart, but the volume occupied by transverse tubules was apparently reduced. The isolated cells had significantly shorter sarcomeres than did cells in the intact tissue, and this was associated with an altered topography of plasma membrane surface folds at the level of the Z-lines. Plasma membrane intramembrane particles were randomly distributed and showed the same numerical density on the E-faces of both isolated and intact-heart myocytes. However, P-face particle density was slightly reduced in the isolated cells. It is concluded that the few differences detected in the isolated cells do not reflect any fundamental derangement of their properties.

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.

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.

The calcium paradox in isolated frog heart: Ringer revisited.

Restoration of a normal calcium concentration in the perfusate of isolated hearts after a short period of calcium-free perfusion may result in irreversible cell damage (calcium paradox). We have compared the calcium paradox in rat and frog hearts. Perfusion with zero calcium for 8 min at 37 degrees C predisposed the rat heart to the paradox. After the reintroduction of calcium to the perfusate resting tension rose, developed tension did not recover, ultrastructural changes occurred and enzyme loss was substantial. In the frog heart a calcium paradox did not occur after 8 min of calcium-free perfusion at 23 degrees C. Removal of both potassium and calcium caused a rise in resting tension on reintroduction of control solution, but the rise was only transient and absent if potassium was present during the perfusion with zero calcium. At 37 degrees C no complete calcium paradox occurred after 8 min calcium-free perfusion. Only a small rise in resting tension was apparent, and developed tension partially recovered. A calcium paradox could only be induced in the frog heart after calcium-free perfusion at 37 degrees C for 30 min. Ultrastructural changes were apparent and resting tension rose but even under these conditions the recovery of developed tension was not abolished. Release of creatine kinase was 161 +/- 55 IU/g dry tissue during the 15 min after reintroduction of calcium (n = 5). Calcium-free perfusion for 8 min resulted in a smaller release of creatine kinase over 15 min (39 +/- 11 IU/g dry tissue. n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

The cardioprotective effect of verapamil.

1. Prolonged periods of normothermic ischaemia and reperfusion produce changes in the ultrastructure of the myocardium. Experiments were undertaken to establish whether the prophylactic use of verapamil provided protection. 2. Rabbits were pretreated with verapamil (2 mg/kg twice daily s.c. for 4-5 days). The hearts were isolated, perfused aerobically, made ischaemic or made ischaemic and then reperfused. The hearts were then perfusion fixed and sectioned for electronmicroscopy. 3. Verapamil pretreatment decreased the ultrastructural damage caused by ischaemia and reperfusion.

The effect of dimethylsulfoxide on the calcium paradox.

Reperfusion of isolated rat hearts with calcium-containing solution after a short period of calcium-free perfusion results in irreversible cell damage (calcium paradox). Experiments were undertaken to study the effect of dimethylsulfoxide (DMSO) on the occurrence of the calcium paradox in rat heart muscle. DMSO (1.4 mol/l) was added to the calcium-free or the reperfusion medium. Cell damage was quantitated in terms of creatine kinase (CK) release, cardiac electrogram (CEG) changes, and ultrastructural damage. Reperfusion with calcium-containing solution without DMSO after calcium-free perfusion with DMSO resulted in massive release of CK, electrical arrest of the ventricles, and formation of contraction bands. Nearly 100% of the cells displayed the calcium paradox damage. Reperfusion with calcium-containing solution with DMSO after calcium-free perfusion without DMSO resulted in a moderate release of CK, and electrical arrest of the ventricles. The myofibrils remained in a relaxed state. Only 4% of the cells displayed the calcium paradox damage. These results indicate that DMSO does not protect rat hearts against the effects of calcium-free perfusion that predispose the myocardium to the calcium paradox. The calcium paradox damage is reduced, however, when DMSO is present during the reperfusion phase.