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Biomedical subjects

W G McLean

Publications and source records attributed to W G McLean.

At least 19 recordsLinked to original sources
PubMed1997

Protein kinase C isozyme expression in sciatic nerves and spinal cords of experimentally diabetic rats.

Changes in the expression and activation of protein kinase C (PKC) have been implicated in the pathogenesis of diabetic neuropathy. Recent studies in liver, retina, and cardiovascular tissues from experimentally diabetic rats have demonstrated that diabetes has a selective effect on the expression and subcellular distribution of isozymes of PKC. In the light of this evidence, we investigated the expression of the PKC isozymes alpha, betaI, betaII, and gamma in sciatic nerves, spinal cords, and in the L4,5 dorsal root ganglia from streptozotocin-induced diabetic rats. Six weeks of diabetes had differential effects on the expression and distribution of PKC isozymes in sciatic nerves and spinal cords. In the sciatic nerves there was an apparent translocation of the alpha isoform from the cytosolic to the particulate fractions, the betaII isoform was reduced in the cytosolic fraction, and the betaI and gamma isoforms were unaffected. The changes in the isozyme immunoreactivities in the nerves were not a direct result of changes in either spinal cord or dorsal root ganglia alone, suggesting that diabetes has different effects on motor and sensory fibres and/or on Schwann cells. In nerves that had been crushed 14 days previously there was an increase in total PKC alpha immunoreactivity. This increase was potentiated in diabetic rats. On the other hand, PKC betaII immunoreactivity in crushed nerves was unaffected by diabetes. The data are consistent with diabetes-induced changes in expression of PKC betaII contributing to nerve damage, and changes in PKC alpha being a consequence of it.

Animals↗
PubMed1997

Enhanced in vitro neurotoxicity of artemisinin derivatives in the presence of haemin.

The role of haem in the neurotoxicity of artemisinin derivatives has been studied in vitro by examining neurite outgrowth measured by image analysis and cellular metabolism of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) measured spectrophotometrically in the neuroblastoma cell line NB2a, and by examining binding of radiolabelled dihydroartemisinin to NB2a cell and rat brain proteins. In the cases of artemether, dihydroartemisinin, and arteether, haemin (ferriprotoporphyrin IX) significantly increased the dose-related inhibition of neurite outgrowth from differentiating NB2a cells and significantly increased the dose-dependent inhibition of MTT metabolism. Inhibition of neurite outgrowth and metabolism of MTT in the presence or absence of haemin ranged from 72% to 93% and from 27% to 49% at a drug concentration of 300 nM. Haemin also significantly increased the dose-related binding of radiolabelled dihydroartemisinin to proteins from NB2a cells approximately twofold and to rat brain between three- and sixfold. Haemin did not enhance the neurotoxicity of desoxyarteether, a structural analogue of arteether with an ether linkage in the place of the endoperoxide bridge. It is suggested that haemin may catalyse the transformation of these derivatives via an interaction with the endoperoxide bridge of the artemisinin derivative to produce free radicals or electrophilic intermediates that are toxic to neuronal cells.

Animals↗
PubMed1996

Altered GAP-43 immunoreactivity in regenerating sciatic nerve of diabetic rats.

Experimental diabetes in the rat is associated with impaired axon regeneration. Successful regeneration depends on the construction of axonal growth cones and establishment of appropriate target connections. The growth-associated protein (GAP)-43 is a major component of the axonal growth cone, and its synthesis and axonal transport are markedly increased during regeneration. The purpose of this study was to determine the effect of experimental diabetes on the synthesis and axonal transport of GAP-43 in regenerating sciatic nerves. Rats were rendered diabetic with 50 mg/kg streptozotocin i.p. Four weeks later, the rats were anesthetized, and one sciatic nerve was crushed to induce regeneration. After 2 weeks, nerves were ligated, and 6 h later, nerve pieces proximal to the ligature and dorsal root ganglia were removed, and proteins were separated by PAGE. Western blots of gels were probed with antibody 10E8/E7 against GAP-43. The presence of GAP-43 was confirmed by immunohistochemistry of nerve sections. Densitometric analysis of the blots showed a 45% reduction in native GAP-43 immunoreactivity in nerve pieces proximal to the ligature (P < 0.05; n = 7). Northern blots of total RNA extracted from pooled dorsal root ganglia were probed with a 32P-radiolabeled cDNA probe for GAP-43. There was no significant difference in the amount of GAP-43 mRNA between diabetic and nondiabetic rats. Immunohistochemistry of sciatic nerve confirmed the reduction in GAP-43 immunoreactivity. We conclude that a defect in turnover or axonal transport of GAP-43 may contribute to the impaired peripheral nerve regeneration in diabetes.

Animals↗
PubMed1995

The toxicity of artemisinin and related compounds on neuronal and glial cells in culture.

The antimalarial drug artemisinin and a number of its derivatives were tested for their effects on proliferation of undifferentiated neuroblastoma Nb2a cells and glioma C6 cells in culture as well as their ability to inhibit neurite outgrowth from Nb2a cells differentiated by removal of serum and addition of dibutyryl cyclic AMP. In the Nb2a and C6 cell cultures, all drugs except desoxyartemisinin significantly inhibited cell proliferation in a dose-related manner with the lowest effective concentration being that of artemisinin at 0.1 microM. Artemether, arteether, artemisinin and dihydroartemisinin also produced a dose-related decrease in the number of neurites/extensions formed by differentiating Nb2a cells, with an effect of dihydroartemisinin at a concentration as low as 1 nM. Desoxyartemisinin had no effect on extension/neurite formation. We propose a potential mechanism for neurotoxicity of artemisinin and its derivatives that involves the endoperoxide bridge which is also known to be necessary for their antimalarial action.

Animals↗
PubMed1994

Loss of the compound action potential: an electrophysiological, biochemical and morphological study of early events in axonal degeneration in the C57BL/Ola mouse.

In the C57BL/Ola (Ola) mouse strain there is a marked slowing of axonal disintegration during Wallerian degeneration. The locus of the mutation controlling this phenomenon (slow Wallerian degeneration--Wlds) has been mapped to chromosome 4, and its protective effect decreases with advancing age. Using biochemical, electrophysiological and histological techniques, the present study was undertaken to determine whether neurofilament phosphorylation and stability are altered or whether calcium-activated proteases are absent in the sciatic nerves of Ola mice. A compound action potential was detectable only when neurofilaments were present and normal axonal architecture was seen. In 1-month-old Ola mice, compound action potentials and neurofilaments were still detectable at 21 days post-transection, whereas both were undetectable by 2 days in BALB/c and C57BL/6J (6J) mice of the same age. Neurofilament levels declined faster with advancing Ola age, confirming previous results, whereas degeneration slowed in ageing BALB/c and 6J mice. In vitro and in vivo degeneration rates were comparable in BALB/c and 6J nerves. Ola nerves, however, showed more rapid decline in vitro than in vivo. Ola and BALB/c nerves frozen and then thawed and incubated in the presence of calcium ions and the ionophore A23187 were not resistant to degradation by intrinsic proteases. Even when a compound action potential could no longer be elicited, however, a majority of nerves still had > 50% of myelinated and unmyelinated axons whose electron microscopic profiles appeared normal. Thus, it appears that the first event in Wallerian degeneration in the Ola mouse is a change at the plasma membrane--a transected nerve becomes unable to conduct a compound action potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
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↗