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M J Cullen

Publications and source records attributed to M J Cullen.

At least 55 records · Page 3Linked to original sources
PubMed1992

The fate of desmin and titin during the degeneration and regeneration of the soleus muscle of the rat.

We studied the fate of desmin and titin in rat skeletal muscle during a cycle of degeneration and regeneration induced in vivo by the inoculation of a snake venom. Cryosections of muscle were labelled using antibodies to the two proteins, and examined at fixed time points after venom injection. Early pathological changes in the muscle, such as hypercontraction, preceded the loss of desmin. Immunolabelling using anti-desmin antibodies showed that desmin bridges were still intact when adjacent myofibrils were no longer aligned. The results suggested that although the hydrolysis of desmin is not necessary for the hypercontraction of muscle fibres, it probably contributes to complete fibre breakdown. Titin, or at least the part which lies close to the M-line, remained intact longer than desmin, but was also hydrolysed prior to complete disintegration of the fibres. Both desmin and titin were re-expressed in the regenerating myotubes by 2 days after venom inoculation, and became well organised even before the myofibrils became aligned. We conclude that desmin and titin are involved in both establishing and maintaining the structural integrity of the muscle fibres.

Animals↗
PubMed1992

The fate of dystrophin during the degeneration and regeneration of the soleus muscle of the rat.

Immunocytochemistry and Western blotting were used to monitor the fate of dystrophin in the soleus muscle of the rat during a cycle of degeneration and regeneration induced by inoculation of the muscle with the venom of Notechis scutatus scutatus (the Australian tiger snake). In control muscle dystrophin was localised close to the plasma membrane. Dystrophin began to break down 3-6 h after venom inoculation, giving a characteristic discontinuous labelling pattern. At 12 h dystrophin was absent from the plasma membrane, and by 1 day the architecture of the muscle fibers had completely broken down. By 2 days post inoculation regeneration had commenced. The regenerating myofibres possessed well-organised myofibrils and the plasma membrane was intact. Dystrophin was detected by Western blot at 3 days, but was not seen in sections until regeneration of the muscle was well advanced, at 4 days post inoculation. The results suggested that although dystrophin was present in the myofibres at 3 days, it was not incorporated into the plasma membrane until 4 days post inoculation. This may be due to the influence of the functional reinnervation of the regenerating fibres, which occurs at 4-5 days, or to the growing fibres reaching a critical diameter.

Animals↗
PubMed1992

The distribution of desmin and titin in normal and dystrophic human muscle.

We have used monoclonal antibodies to desmin and titin, and a combination of immunofluorescence and immunogold labelling to study the disposition of these two proteins in normal human muscle fibres and in fibres at various stages of degeneration in dystrophic muscle. The normal pattern of desmin labelling, in particular the subsarcolemmal labelling, became disrupted at an early stage of fibre breakdown. There was a change from a transverse to a longitudinal orientation of the labelled intermediate filaments as the myofibrils sheared relative to one another. Thus, while it is probable that the desmin filaments are able to play a role in the mechanical integration of the myofibrils in healthy muscle, our results suggest that they cannot withstand the excessive forces generated by the hypercontraction and stretching of dystrophic muscle. However, small accumulations of desmin persisted between the damaged myofibrils until necrosis reached an advanced stage. In general, the degradation of titin appeared to occur before the degradation of desmin, and at the ultrastructural level, labelling with antibodies to epitopes from parts of the titin molecule close to the A-I-band junction was lost before labelling with an antibody to an epitope in the A-band. This suggests that different regions of the titin molecule break down at different stages in the breakdown of the fibre. We propose that lysis of titin in the I-band may underlie 'slippage', an abnormality often seen in dystrophic muscle, in which the A-band slips to one pole of the sarcomere such that it abuts onto the Z-line. Breakdown of the A-band section of titin may facilitate the disassembly of the A-filaments.

Connectin↗
PubMed1992

Imaging of medullary carcinoma of the thyroid using 111In-labelled anti-CEA monoclonal antibody fragments.

The distribution of 111In-labelled anti-carcinoembryonic antigen (CEA) monoclonal antibody fragments [F(Ab')2] was studied in five patients either with known inoperable medullary carcinoma of the thyroid (MCT) or evidence of recurrence/metastases based on elevated calcitonin (hCT) levels. All five cases had elevated serum CEA levels and positive immunohistochemical stains for both hCT and CEA prior to scintigraphy. In two patients with identified inoperable disease both planar and SPECT scans were positive. In the remaining three patients, where the recurrence/metastatic sites were unknown, SPECT images were positive in two. Of these, only one had positive planar images. These results indicate that 111In-labelled anti-CEA F(ab')2 scintigraphy, especially in conjunction with SPECT, is useful for the diagnostic evaluation of patients with MCT. The limiting factor of this technique is the high level of non-specific uptake, particularly in the liver, but improvements in the specificity of newer anti-CEA antibodies and the ability to label these with 99Tcm is addressing this problem.

Adult↗
PubMed1992

Behavioral effects of A71623, a highly selective CCK-A agonist tetrapeptide.

We studied the behavioral effects of a novel cholecystokinin tetrapeptide (CCK-4) analogue, A71623, with full agonist activity and high affinity and selectivity for the CCK-A receptor subtype relative to the CCK-B receptor. In tests for anorectic activity, A71623 was found to suppress 60-min intakes of a liquid diet in both deprived and sated rats, and the effects were blocked by a selective CCK-A antagonist, A70104. Compared with CCK-8, A71623 was found to have improved potency and duration of action; the most potent route of administration was intraperitoneal. A71623 also suppressed the intake of a liquid diet and a 0.2 M sucrose solution in lean and obese Zucker rats. In daily injection studies, the anorectic activity of CCK-8 diminished rapidly, whereas the suppressant effects of A71623 on food intakes and body weight gains persisted throughout the 11-day treatment period. Finally, A71623 reduced the spontaneous locomotor activity of rats at doses above those required to suppress intakes. These studies are the first to describe the behavioral effects of a potent and highly selective CCK-A receptor agonist.

Animals↗
PubMed1991

The effect of denervation on the morphology of regenerating rat soleus muscles.

This study examines the level to which muscle regeneration proceeds in the absence of innervation. Regeneration was monitored in rat soleus muscles following localised injection of a snake toxin, notexin. Muscles which had been concomittantly denervated were compared with those that were normally innervated. Until 3-4 days following toxin administration regeneration is identical in both groups. The muscles contain new myotubes in place of the degenerated "parent" fibres. Thereafter, the non-denervated muscles grow rapidly and by 28 days their myofibres attain the size of those from the contralateral controls. Growth of denervated regenerating muscles, however, is retarded and is superseded by a gradual atrophy. In such muscles we further identify ultrastructural abnormalities from 7 days post-injection. These a re loss of individual myosin filaments and the presence of immature and abnormal configurations of the transverse system and triads. We, thus, conclude that innervation is an obligatory requirement for the restoration of normal myofibrillar and sarcotubular morphology, as well as growth, but is not necessary for the neo-formation of myofibres.

Animals↗
PubMed1991

Immunogold labelling of dystrophin in human muscle, using an antibody to the last 17 amino acids of the C-terminus.

Immunolabelling with a 10 nm gold probe was used to localize dystrophin at the ultrastructural level in human skeletal muscle. The primary antibody was raised against a synthetic peptide containing the last 17 amino acids at the C-terminus of dystrophin. Using this antibody, labelling was almost entirely confined to a narrow band enclosing 40 nm either side of the plasma membrane and including the membrane itself. Histograms of the position of the gold probe relative to the plasma membrane showed modes lying over the membrane itself or the extracellular face of the membrane. One interpretation of these results is that the C-terminus of dystrophin is inserted in the plasma membrane alongside the glycoproteins with which it is tightly associated. Histograms of the distances between gold probes displayed modes at approximately 120 nm in both transverse and longitudinal sections suggesting that dystrophin forms a lattice-like network adjacent to the plasma membrane.

Amino Acid Sequence↗
PubMed1991

Myopathic changes in indirectly stimulated mouse diaphragm after ecothiopate in vitro.

Mouse phrenic nerve-hemidiaphragms were stimulated in vitro in the presence of the anticholinesterase ecothiopate iodide and prepared for light and electron microscopy at different times during and after the appearance of prolonged contractions localized at the endplate. The earliest changes were at the subsynapse, without damage to the plasma membrane, and comprised hypercontraction of the sarcomeres, dilatation and vesiculation of the sarcoplasmic reticulum and the mitochondria, and dissolution of the Z-lines. Later there was damage to the plasma membrane. Also appearing later in the junctional region, but separated from the subsynapse by apparently normal muscle, were extrasynaptic hypercontractions, with a plasma membrane initially undamaged, but which became permeable after the contractile material divided into contraction clots. A hypothesis is proposed for the formation of such hypercontractions by abnormal mechanical factors arising from different contractile states along the length of the fibre, and is discussed with the role of prolonged transmitter action in the aetiology of myopathy.

Animals↗
PubMed1990

Ultrastructural localization of dystrophin in human muscle by using gold immunolabelling.

Immunolabelling with a 5 nm gold probe was used to localize dystrophin at the ultrastructural level in human muscle. The primary antibody was monoclonal, raised against a segment (amino acids 1181-1388) from the rod domain of dystrophin. The antibody (Dy4/6D3) is specific for dystrophin and shows no immunoreactivity with any protein from mdx mouse muscle or from patients with a gene deletion spanning part of the molecule recognized by the antibody (Nicholson et al. 1989 a; England et al. 1990). Using this antibody, labelling was almost entirely confined to a narrow 75 nm rim at the periphery of the muscle fibres. Histograms of the distance from the gold probe to the cytoplasmic face of the plasma membrane and of the distance between gold probes (nearest neighbour in a plane parallel with the plasma membrane) displayed modes at approximately 15 nm and 120 nm, respectively. The distribution of the probe was the same in longitudinal and transverse sections of the muscle. These observations suggest that the rod portion of the dystrophin molecule is normally arranged close to the cytoplasmic face of the plasma membrane and that the molecules form an interconnecting network. Labelling was not associated with the transverse tubular system.

Cell Membrane↗
PubMed1990

Charge movements and transverse tubular ultrastructure in organ cultured skeletal muscle.

A study was made of charge movements and the transverse tubular systems in rat EDL and soleus muscle fibres maintained for up to five days in organ culture. In the cultured EDL muscle the maximum amount of charge moved was about one third of that in innervated muscle. Charge movements in innervated soleus fibres are small, less than 10 nC/microF, and difficult to resolve. They remain small following organ culturing. The ultrastructural study examined the concentration of junctional feet because of their proposed key role in excitation-contraction coupling. The general architecture of the triads and the spacing of the feet in both muscle types was largely unchanged by culturing. In cultured EDL muscles the small changes in feet concentration did not parallel the large fall in charge movement. The results reported here support a previous conclusion that, in mammalian muscle, there is not a simple relation between charge and feet. The stimulation of cultured soleus muscles with a fast twitch pattern of electrical activity produced no observable changes in morphology.

Animals↗
PubMed1990

Muscle necrosis caused by snake venoms and toxins.

Snake bite is often associated with localised soft tissue necrosis. Less frequently victims may suffer extensive muscle damage leading to rhabdomyolysis and the loss of muscle-specific protein. This review describes the organisation and structure of mammalian skeletal muscle, and its response to myotoxic venoms and to isolated pure myotoxic venom fractions. The clinical reports of muscle damage in man following snake bite are discussed, and the various classes of myotoxic toxins are introduced. Muscle damage caused by the toxins is next described, particular emphasis being placed on the correlation between muscle pathology seen at the light level and the morphological changes seen at the level of the electron microscope. Where known, those subcellular components of the muscle fibre that are especially sensitive to assault, and those components that appear to be spared, are identified. The relevance of the selective sparing of some components to the regenerative capacity of the skeletal muscle is considered.

Animals↗
PubMed1990

Bradykinin has no acute effect on the response of forearm blood flow to sympathetic stimulation in man.

1. Six healthy male subjects received 0.9% (w/v) NaCl (saline) followed by incremental doses of bradykinin (1, 3 and 10 pmol/min), via the left brachial artery. Blood flow and the response of blood flow to lower-body negative pressure were measured in both forearms during infusion of saline and each dose of bradykinin. 2. Bradykinin produced a moderate and dose-dependent increase in blood flow in the infused, but not the non-infused, forearm. Lower-body negative pressure produced an approximately 15-20% reduction in blood flow in both forearms, and this response was unaffected by local infusion of bradykinin. 3. Bradykinin, in contrast to angiotensin II, had no acute effect on peripheral sympathetic responses to lower-body negative pressure. We conclude that, in forearm resistance vessels in man, withdrawal of angiotensin II, rather than accumulation of bradykinin, is likely to account for the attenuation of peripheral sympathetic responses after acute administration of a converting-enzyme inhibitor.

Adult↗
PubMed1990

Severe anaphylactoid reaction to hydroxyethyl starch.

A case is presented of a severe anaphylactoid reaction to hydroxyethyl starch solution that occurred peri-operatively and required extended intensive management of the resultant bronchospasm and hypotension. Subsequent intradermal injection of hetastarch produced a delayed positive response, suggestive of a complement-mediated mechanism.

Aged↗
PubMed1989

Inhibition of protein synthesis during CNS myelination produces focal accumulations of membrane vesicles in oligodendrocytes.

Optic nerves of Xenopus tadpoles were exposed to cycloheximide to identify changes that occur during CNS myelin membrane formation when protein synthesis is inhibited. Groups of stage 51-56 tadpoles were immersed in either 10 or 20 micrograms ml-1 cycloheximide, and at specified times between 12 and 18 h after initial immersion tadpoles were killed and their optic nerves prepared for ultrastructural analysis. As early as 12 h there were alterations in oligodendrocytes from treated animals compared with control animals. The number of polyribosomes in the perikarya and cell processes was greatly reduced and the rough endoplasmic reticulum was disorganized. Mitochondria and microtubules were normal in appearance. Many oligodendroglial tongue processes at the inner margin of the myelin sheath were enlarged, occasionally indented the axon and were filled with vesicular profiles. Vesicles were noted in other cytoplasmic regions of oligodendrocytes and focal changes in the lamellar structure of myelin were found in paranodal regions. The internodal portions of the myelin sheath, axons and astrocytes appeared normal. Polyacrylamide gels of optic nerves showed that the incorporation of 35S-methionine into polypeptides had been almost completely inhibited by treatment with cycloheximide. These observations suggest that cycloheximide, by inhibiting synthesis of myelin proteins, alters the ability of oligodendrocytes to incorporate membrane components into CNS myelin sheaths.

Animals↗
PubMed1989

Centrally administered CCK-8 suppresses activity in mice by a "peripheral-type" CCK receptor.

Cholecystokinin octapeptide (CCK-8) administered either systemically (IP) or centrally (ICV) suppresses several types of behavior in mice including exploratory locomotion, rearing and grooming. At doses equimolar to those active for CCK-8, neither desulfated CCK-8 (CCK-8-DS), nor the protected C-terminus tetrapeptide fragment, BOC-CCK-4, is behaviorally active when administered either centrally or systemically. A potent and selective antagonist to the peripheral type (Type A) CCK receptor, A-65186, when given systemically, blocked the effects of systemically administered CCK-8, but failed to block the effects of ICV administered CCK-8. Central administration of A-65186 blocked the effects of ICV administered CCK-8. These results demonstrate that administration of exogenous CCK-8 to mice can suppress exploratory locomotion by acting either centrally or peripherally and that in either case the demonstrated behavioral effects are mediated via a "peripheral" type (Type A) CCK receptor.

Animals↗
PubMed1988

An isotopic rubidium ion efflux assay for the functional characterization of nicotinic acetylcholine receptors on clonal cell lines.

An isotopic rubidium ion efflux assay has been developed for the functional characterization of nicotinic acetylcholine receptors on cultured neurons. This assay first involves the intracellular sequestration of isotopic potassium ion analog by the ouabain-sensitive action of a sodium-potassium ATPase. Subsequently, the release of isotopic rubidium ion through nicotinic acetylcholine receptor-coupled monovalent cation channels is activated by application of nicotinic agonists. Specificity of receptor-mediated efflux is demonstrated by its sensitivity to blockade by nicotinic, but not muscarinic, antagonists. The time course of agonist-mediated efflux, within the temporal limitations of the assay, indicates a slow inactivation of receptor function on prolonged exposure to agonist. Dose-response profiles (i) have characteristic shapes for different nicotinic agonists, (ii) are described by three operationally defined parameters, and (iii) reflect different affinities of agonists for binding sites that control receptor activation and functional inhibition. The rubidium ion efflux assay provides fewer hazards but greater sensitivity and resolution than isotopic sodium or rubidium ion influx assays for functional nicotinic receptors.

Animals↗