Search PubMed⌕ Search

Biomedical subjects

W G McLean

Publications and source records attributed to W G McLean.

At least 37 records · Page 2Linked to original sources
PubMed1994

The disruption of brain microtubules in vitro by the phospholipase inhibitor p-bromophenacyl bromide.

The influence of p-bromophenacyl bromide (pBPAB) and structural analogues on the assembly and Ca2+ sensitivity of porcine brain microtubules (MTs) was studied by spectrophotometric measurements in vitro. MT assembly was inhibited by 36 microM pBPAB but not by the structural analogues p-chlorophenacyl chloride or acetophenone. In the presence of pBPAB, but not structural analogues, the addition of 10 mM Ca2+ induced aggregation of polymerized MT protein, whereas a decrease in turbidity (due to MT disassembly) was observed in controls. The effects of pBPAB on both MT assembly and Ca2+ sensitivity were blocked by glutathione, but not by N-acetyl L-cysteine, N-acetyl L-lysine nor L-tyrosine, indicating that a highly reduced sulphydryl group(s) may be involved. Western blotting analyses of drug-treated MTs revealed a form of tubulin with altered electrophoretic characteristics, probably caused by a covalent interaction with pBPAB. MT preparations polymerized in the presence of the drug contained fewer MTs than control samples, the predominant structures being identified as amorphous aggregates of MT proteins. The fact that pBPAB affects MT integrity at an effective anti-inflammatory dose in vitro may reflect the involvement of MT disruption in some of the pharmacological effects of this drug. pBPAB is not therefore a suitable tool for studying the specific involvement of phospholipase A2 in cellular events.

Acetophenones↗
PubMed1994

The toxicity of organophosphate compounds towards cultured PC12 cells.

The effects of three representative organophosphates (OPs), tricresyl phosphate (TCP), triphenyl phosphite (TPP) and paraoxon (POX) on the proliferation and viability of rat PC12 pheochromocytoma cells were studied. With respect to its IC50, TCP was at least an order of magnitude more potent in its antiproliferative activity than both TPP and POX. All test OPs were cytotoxic at concentrations inhibiting cell proliferation. No compound inhibited cell growth below 10 micrograms/ml. For TCP and TPP the estimated IC50 values from proliferation assays were lower than published LD50 values in vivo, whereas paraoxon was much less toxic in vitro than in vivo. Subcytotoxic levels of TCP (1 micrograms/ml) were found to inhibit the maintenance of neurites on cells grown in the presence of nerve growth factor.

Animals↗
PubMed1994

Tyrosination state of alpha-tubulin in regenerating peripheral nerve.

Certain modifications of the neuronal cytoskeleton that are associated with development also occur during regeneration of adult mammalian peripheral nerve. The aim of the present study was to examine one such modification, the tyrosination of alpha-tubulin. Adult rats were anaesthetized and the left or right sciatic nerve randomly selected and crushed to induce regeneration. In certain instances nerves were crushed then ligatured about the crush, to prevent regeneration. Five days later the rats were killed and the regenerating (or ligatured) and the contralateral (control) nerves were removed. Quantitative immunoblotting of nerve homogenates with antibodies that recognize tyrosinated alpha-tubulin and total alpha-tubulin revealed a significant increase (p < 0.01) in the proportion of alpha-tubulin that was tyrosinated in nerve pieces distal (peripheral) to a nerve crush and to uncrushed nerve. No such difference occurred in ligatured (crushed but nonregenerating) nerve, implying that the increase was related to the presence of regenerating fibres; nor was there any gradient in tyrosination of alpha-tubulin in control nerves. This effect was confirmed by cytofluorimetric scanning and fluorescence confocal laser scanning microscopy of fixed sections of control and regenerating nerve, stained with antibodies directed against tyrosinated alpha-tubulin. When nerves were separated into fractions containing assembled and nonassembled tubulin, a significant (p < 0.01) increase was found in the proportion of tyrosinated alpha-tubulin in the nonassembled tubulin fraction in nerve pieces containing regenerating fibres. This occurred in the absence of a change in the proportion of assembled and nonassembled tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Changes in slow axonal transport of tubulin induced by local application of colchicine to rabbit vagus nerve.

The biochemical and morphological responses of the rabbit vagus nerve to local application of colchicine and to nerve crush were investigated. Fourteen days after the cervical vagus nerve had been crushed or subjected to local application of colchicine for 2 h, nodose ganglia of anaesthetized rabbits were either injected with [35S]methionine or [3H]leucine for studies of slow and fast axonal transport, respectively, or prepared for light microscopical examination. The radio-labelled proteins of the faster of the two slow transport groups (SCb; 25-30 mm day-1) were separated by one- or two-dimensional polyacrylamide gel electrophoresis and both radio-labelled tubulin and actin were quantified by densitometry from resulting fluorographs of gels. A relative increase in radio-labelled tubulin was found in SCb in the crushed and colchicine-treated nerves; this increase persisted for up to 50 days after nerve crush. Morphological changes in nerve cell bodies induced by colchicine were similar, but smaller in magnitude than those in crushed nerves. It is concluded that a temporary arrest of axonal transport produced by colchicine can lead to a redistribution of tubulin transport comparable with that found in regenerating nerve.

Animals↗
PubMed1993

Subcellular distribution and immunological detection of retrograde axonally transported proteins in acrylamide and diabetic neuropathies.

Neuropathies produced by both acrylamide- and streptozotocin-induced diabetes in the rat are accompanied by a deficit in the retrograde axonal transport of a defined group of proteins that can be visualized on two-dimensional polyacrylamide gels. In this work, these proteins are identified as being primarily soluble and being absent from rat brain. They are not immunologically related to the major retrogradely transported protein synaptophysin. Polyclonal antiserum to the proteins was produced in mice and used to confirm the reduction in their retrograde transport in sciatic nerve of diabetic and acrylamide-treated rats.

Acrylamide↗
PubMed1993

An in vitro system for the study of slow axonal transport.

Here we present a model system which for the first time permits studies of slow axonal transport in vitro. Axonally transported proteins of rat vagus nerves were radiolabelled with [35S]methionine in the nodose ganglion in vitro and were incubated for up to 3 days in culture medium. Slowly transported proteins were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis and identified on Western blots of two-dimensional gels with antibodies to actin and alpha-tubulin. The system will be valuable for pharmacological analysis of the mechanisms of slow transport.

Animals↗
PubMed1993

Axonal transport and morphological changes following nerve compression. An experimental study in the rabbit vagus nerve.

Axonal transport and morphological changes were studied in the rabbit vagus nerve after the nerves had been subjected to compression at either 0, 50 or 200 mmHg for two hours. Slow axonally transported proteins, tubulin and actin, were radiolabelled with 35S-methionine two, seven or 14 days after the injury and the distribution of radiolabelled tubulin and actin within component b of slow transport was measured three days later by densitometric analysis of fluorographs of polyacrylamide gel. No significant differences were found in the distribution of tubulin two (50 and 200 mmHg) or seven (200 mmHg) days after injury, but at 14 days (200 mmHg) there was significantly increased radiolabelling of tubulin relative to actin in the nerve 60 to 70 mm from the nodose ganglion. Morphometric measurements of the nerve cell bodies two days after the compression injury at 200 mmHg revealed no significant changes. Previous work has shown that morphological changes, similar to those found after axotomy, were present in nerve cell bodies seven days after a compression injury. This, taken together with the present results, indicates that compression can induce both morphological and biochemical changes in the neurone. The altered axonal transport of tubulin associated with nerve injury follows a slower time course and does not precede the morphological changes. The findings may be of relevance when discussing the double crush syndrome.

Actins↗
PubMed1993

Glycation of brain actin in experimental diabetes.

Actin is a neuronal protein involved in axonal transport and nerve regeneration, both of which are known to be impaired in experimental diabetes. To determine if actin is subject to glycation, we rendered rats diabetic by injection of streptozotocin. Two or 6 weeks later brains were removed and a preparation of cytoskeletal proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis. Brains from diabetic animals contained an extra polypeptide that migrated close to actin and reacted with monoclonal antibody C4 against actin. It was also found in a preparation of soluble synaptic proteins from diabetic rat brain, indicating that it was at least partly neuronal in origin. This polypeptide could be produced by incubation of cytoskeletal proteins from brains of nondiabetic rats with glucose-6-phosphate in vitro. The appearance of this glycated actin in diabetic animals was prevented by administration of insulin for a period of 6 weeks. We could not detect any effect of glycation in vitro on the ability of muscle G-actin to form F-actin filaments and its significance for the function of actin remains to be determined. The finding that glycation of platelet-derived actin from diabetic patients was significantly increased implies that the abnormality may also occur in clinical diabetes.

Actins↗
PubMed1991

Glycation of rat sciatic nerve tubulin in experimental diabetes mellitus.

Diabetic neuropathy is associated with some early defects of axonal transport in experimental animals. Axonal transport is dependent on intact microtubules, and unsubstituted lysine residues of tubulin are essential for microtubule polymerization. As lysine residues are the major target for the non-enzymatic attachment of glucose, the effect of diabetes on the extent of glycation of tubulin was investigated. There was a more than four-fold increase in the extent of glycation of tubulin in the sciatic nerve of rats with streptozotocin-induced diabetes of 2 weeks duration compared with control rats. In contrast, no such increase in glycation was observed in brain microtubule protein from diabetic rats at that stage of diabetes. Incubation of brain microtubule protein with glucose prior to in vitro polymerization showed that the early stages of glycation were not associated with inhibition of microtubule assembly. The observed glycation of peripheral nerve tubulin in early experimental diabetes may nevertheless contribute to axonal transport abnormalities through an as yet undetermined impairment of microtubule function.

Animals↗
PubMed1991

Antibodies to tubulin and microtubule-associated proteins. A study in diabetes mellitus, systemic lupus erythematosus, and rheumatoid arthritis.

We report the results of a study of serum antibodies to proteins of the nerve cytoskeleton in patients with Type I and Type II diabetes mellitus, both with and without clinical signs of diabetic neuropathy. In contrast to previous reports, elevated levels of antibody to tubulin or glycated tubulin were not associated with either diabetes or diabetes with related neuropathy. Similarly, clinical evidence of neuropathy in patients with diabetes did not relate to increased levels of antibody to native or glycated microtubule-associated proteins (MAPs). The levels of antibody to MAPs and glycated MAPs were higher in control subjects over the age of 45 years compared with younger control subjects. Increased levels of antibody to tubulin and glycated tubulin were found in the sera of patients with systemic lupus erythematosus, but not rheumatoid arthritis.

Adult↗
PubMed1991

Neurofilament protein phosphorylation in spinal cord of experimentally diabetic rats.

This study was designed to determine if the known decrease in slow axonal transport of proteins in the sciatic nerve of experimentally diabetic rats is related to altered phosphorylation of neurofilament proteins (NFPs). Rats were rendered diabetic with 50 mg/kg of streptozotocin, i.p. At 3 and 6 weeks later, NFPs were prepared from spinal cord. The in vivo phosphorylation state of NFPs was examined by using phosphate-dependent (RT97) and -independent (RMd09) antibodies against high-molecular-mass NFPs on Western blots. Neurofilament-associated kinase activity was also measured in vitro by incubation of NFPs with [32P]ATP. Phosphorylation of all three NFPs (high, medium, and low molecular mass) occurred, as confirmed by gel electrophoresis and autoradiography. At 30 min of incubation, protein-bound radioactivity in NFPs from diabetic animals was reduced to 86.7 +/- 3.4 and 54.3 +/- 19.6% of that in nondiabetic animals at 3 and 6 weeks of diabetes, respectively (p less than 0.001 and p less than 0.05, respectively). NFPs were also incubated with acid phosphatase and rephosphorylated. Results showed that the increased in vivo phosphorylation contributed to the decreased in vitro phosphorylation. Extraction of protein kinases and addition back to the NFPs revealed, in addition, a reduced activity in the diabetic animals of the protein kinases measured in vitro.

Animals↗
PubMed1990

The effect of acrylamide on the induction of ornithine decarboxylase in the dorsal root ganglion of the rat.

Injury of the rat sciatic nerve is accompanied by an increased activity of the enzyme ornithine decarboxylase (ODC) in dorsal root ganglia. This increase is impaired in streptozotocin-induced diabetes, in which retrograde axonal transport of proteins is reduced. In order to confirm the relationship between altered axonal transport and ODC induction we treated rats with acrylamide i.p. to cumulative doses of 150 and 350 mg/kg. One sciatic nerve was crushed under anaesthesia and 24 h later dorsal root ganglia were removed and assayed for ODC activity by a dual-label radioenzymatic method. The ratio of activity of 2.41 +/- 0.57 (crushed side over control side) was reduced to 1.66 +/- 0.9 and 1.7 +/- 0.65 after acrylamide treatment at 150 and 350 mg/kg, respectively. The results are consistent with the postulated role of retrograde axonal transport in the cell body responses to nerve injury and may explain the effect of acrylamide on nerve regeneration.

Acrylamide↗
PubMed1990

The effect of 2,5-hexanedione on the induction of ornithine decarboxylase in the dorsal root ganglion of the rat.

Rat dorsal root ganglia respond to sciatic nerve injury with an increase in the activity of the enzyme ornithine decarboxylase (ODC). The increase is impaired under certain conditions (e.g. diabetes, Vinca alkaloid treatment) where retrograde axonal transport is reduced. The purpose of the experiments was to determine if the neurotoxin 2,5-hexanedione, also known to interfere with retrograde axonal transport, similarly affected ODC induction. Rats were treated with 2,5-hexanedione i.p. to a cumulative dose of 6 and 8 g/kg. One sciatic nerve was crushed under anaesthesia and 24 h later the dorsal root ganglia were removed and assayed for ODC activity by a radioenzymatic method. The ratio of ODC activity of 1.57 +/- 0.58 (crushed side over control side) was reduced to 1.02 +/- 0.41 1.08 +/- 0.39 after 2,5-hexanedione at 6 g and 8 g/kg, respectively. The enzyme was not inhibited by addition of 2,5-hexanedione in vitro. The results confirm the role of retrograde axonal transport in nerve cell responses to injury and are consistent with the effects of 2,5-hexanedione on nerve regeneration.

Animals↗