Comparing the fatty acid composition of organic and conventional milk.

During a 12-mo longitudinal study, bulk-tank milk was collected each month from organic (n = 17) and conventional (n = 19) dairy farms in the United Kingdom. All milk samples were analyzed for fatty acid (FA) content, with the farming system type, herd production level, and nutritional factors affecting the FA composition investigated by use of mixed model analyses. Models were constructed for saturated fatty acids, the ratio of polyunsaturated fatty acids (PUFA) to monounsaturated fatty acids, total n-3 FA, total n-6 FA, conjugated linoleic acid, and vaccenic acid. The ratio of n-6:n-3 FA in both organic and conventional milk was also compared. Organic milk had a higher proportion of PUFA to monounsaturated fatty acids and of n-3 FA than conventional milk, and contained a consistently lower n-6:n-3 FA ratio (which is considered beneficial) compared with conventional milk. There was no difference between organic and conventional milk with respect to the proportion of conjugated linoleic acid or vaccenic acid. A number of factors other than farming system were identified which affected milk FA content including month of year, herd average milk yield, breed type, use of a total mixed ration, and access to fresh grazing. Thus, organic dairy farms in the United Kingdom produce milk with a higher PUFA content, particularly n-3 FA, throughout the year. However, knowledge of the effects of season, access to fresh grazing, or use of specific silage types could be used by producers to enhance the content of beneficial FA in milk.

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.

Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes.

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with 3H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of 32P into purified neurofilament proteins when they were incubated with 32P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.

Synthesis and antimalarial activity of sixteen dispiro-1,2,4, 5-tetraoxanes: alkyl-substituted 7,8,15,16-tetraoxadispiro[5.2.5. 2]hexadecanes.

Sixteen alkyl-substituted dispiro-1,2,4,5-tetraoxanes (7,8,15, 16-tetraoxadispiro[5.2.5.2]hexadecanes) were synthesized to explore dispiro-1,2,4,5-tetraoxane SAR and to identify tetraoxanes with better oral antimalarial activity than prototype tetraoxane 1 (WR 148999). The tetraoxanes were prepared either by peroxidation of the corresponding cyclohexanone derivatives in H(2)SO(4)/CH(3)CN or by ozonolysis of the corresponding cyclohexanone methyl oximes. Those tetraoxanes with alkyl substituents at the 1 and 10 positions were formed as single stereoisomers, whereas the five tetraoxanes formed without the stereochemical control provided by alkyl groups at the 1 and 10 positions were isolated as mixtures of diastereomers. Three of the sixteen tetraoxanes were inactive (IC(50)'s > 1000 nM), but five (2, 6, 10, 11, 12) had IC(50)'s between 10 and 30 nM against the chloroquine-sensitive D6 and chloroquine-resistant W2 clones of Plasmodium falciparum compared to corresponding IC(50)'s of 55 and 32 nM for 1 and 8.4 and 7.3 nM for artemisinin. We suggest that tetraoxanes 13, 16, and 17 were inactive and tetraoxanes 4 and 7 were weakly active due to steric effects preventing or hindering peroxide bond access to parasite heme. Tetraoxanes 1, 10, 11, and 14, along with artemisinin and arteether as controls, were administered po b.i.d. (128 mg/kg/day) to P. berghei-infected mice on days 3, 4, and 5 post-infection. At this dose, tetraoxanes 10, 11, and 14 cured between 40% and 60% of the infected animals. In comparison, artemisinin and tetraoxane 1 produced no cures, whereas arteether cured 100% of the infected animals. There was no apparent relationship between tetraoxane structure and in vitro neurotoxicity, nor was there any correlation between antimalarial activity and neurotoxicity for these seventeen tetraoxanes.

The effect of nerve stimulation on the axonal transport of noradrenaline and dopamine-beta-hydroxylase.

1 A method for stimulating the lumbar sympathetic outflow from the spinal cord of the rat is described which does not require artificial respiration of the animal.2 In some, but not all experiments continuous stimulation at 2 Hz or intermittently at 10 Hz accelerated the rate at which noradrenaline and dopamine-beta-hydroxylase accumulated central to a ligature on the sciatic nerve by approximately 40%.3 It is concluded that, although nervous activity is not necessary for axonal transport of transmitter granules in sympathetic neurones, intense nervous activity may accelerate the rate of granule transport.

Impaired induction of nerve ornithine decarboxylase activity in the streptozotocin-diabetic rat is prevented by the aldose reductase inhibitor ponalrestat.

1. The present study was designed to investigate if the aldose reductase inhibitor ponalrestat is capable of preventing the impairment of the response of ornithine decarboxylase (ODC) to nerve crush in streptozotocin (STZ)-diabetic rats. 2. ODC activity was measured in the dorsal root ganglia of crushed and uncrushed contralateral sciatic nerve of non-diabetic, ponalrestat-treated non-diabetic, STZ-diabetic and ponalrestat-treated STZ-diabetic rats. 3. Twenty four hours after crush, a significant (P less than 0.001) increase in the ratio of ODC activity in ganglia of crushed relative to uncrushed nerves was found in non-diabetic but not in diabetic rats, as expected. In the ponalrestat-treated diabetic rats the ratio was significantly higher (P less than 0.001) than that in the untreated diabetic rats and was not different from that in the non-diabetic group. 4. Ponalrestat also significantly decreased absolute levels of ODC activity in ganglia of uncrushed nerves from diabetic and non-diabetic animals. Despite the near-normal induction of ODC activity by nerve crush in the ponalrestat-treated diabetic animals, absolute ODC activity remained lower than that in ganglia of uncrushed nerves from non-diabetics. 5. We conclude that ponalrestat is able to prevent the impaired induction of ODC in experimental diabetes. The results, however, call into question the relationship between impaired ODC induction and diabetes-induced defects in nerve regeneration, which are insensitive to ponalrestat.

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.

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.

The role of glutathione in the neurotoxicity of artemisinin derivatives in vitro.

The role of antioxidants in the neurotoxicity of the antimalarial endoperoxides artemether and dihydroartemisinin was studied in vitro by quantitative image analysis of neurite outgrowth in the neuroblastoma cell line NB2a. Intracellular glutathione concentrations were measured by high performance liquid chromatography with fluorescence detection. Both dihydroartemisinin (1 microM) and a combination of artemether (0.3 microM) plus haemin (2 microM) significantly inhibited neurite outgrowth from differentiating NB2a cells to 11.5 +/- 11.0% (SD) and 19.6 +/- 15.2% of controls, respectively. The inhibition by artemether/haemin was prevented by the antioxidants superoxide dismutase (109.7 +/- 47.8% of control), catalase (107.0 +/- 29.3%) glutathione (123.8 +/- 12.4%), L-cysteine (88.0 +/- 6.3%), N-acetyl-L-cysteine (107.8 +/- 14.9%), and ascorbic acid (104.3 +/- 12.7%). Dihydroartemisinin-induced neurotoxicity was completely or partially prevented by L-cysteine (99.5 +/- 17.7% of control), glutathione (57.9 +/- 23.4% of control), and N-acetyl-L-cysteine (57.3 +/- 9.5%), but was not prevented by superoxide dismutase, catalase, or ascorbic acid. Buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase, significantly increased the neurotoxic effect of non-toxic concentrations of artemether/haemin (0.1 microM/2 microM) and dihydroartemisinin (0.2 microM), suggesting that endogenous glutathione participates in the prevention of the neurotoxicity of artemether/haemin and dihydroartemisinin. Artemether/haemin completely depleted intracellular glutathione levels, whereas dihydroartemisinin had no effect. We conclude that although glutathione status is an important determinant in the neurotoxicity of endoperoxides, depletion of glutathione is not a prerequisite for their toxicity. This is consistent with their mechanisms of toxicity being free radical-mediated damage to redox-sensitive proteins essential for neurite outgrowth, or alteration of a redox-sensitive signalling system which regulates neurite outgrowth.

Axonal transport of actin and regeneration rate in non-myelinated sensory nerve fibres.

The relationship was examined between the rate of regeneration and rate of axonal transport of actin in the sensory fibres of the rabbit vagus nerve. Regeneration rate, determined as the distance moved by [35S]methionine-radiolabelled, fast-transported proteins beyond a crush, was about 3 mm/day. The rate of transport of actin, identified by two-dimensional polyacrylamide gel electrophoresis with fluorography, and DNase affinity chromatography, was 25-30 mm/day. No slower rate of actin transport comparable with regeneration rate, could be found in either control or regenerating nerves. While the provision of actin, by slow axonal transport, to the axonal growth cone may be essential for nerve regeneration, the regeneration rate is not directly controlled by the rate of actin transport.

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.

Cytoskeletal protein mRNA expression in the chick utricle after treatment in vitro with aminoglycoside antibiotics: effects of insulin, iron chelators and cyclic nucleotides.

In birds, spontaneous recovery of the hair cells of the inner ear can occur after damage induced by aminoglycoside antibiotics. The factors that influence this recovery and the process of hair cell regeneration itself have until recently been investigated largely by morphological and histological methods. The aim of this work was to use a molecular biological approach to the analysis of hair cell regeneration by measuring the changes that occur in expression of mRNA for hair cell-specific cytoskeletal proteins fimbrin and class III beta-tubulin, along with that for beta-actin, in the utricle of chicks after hair cell damage both in vitro and in vivo. Utricles were removed from 1-day-old chicks and incubated with the aminoglycoside antibiotics gentamicin or neomycin (both 1 mM), or chicks were injected intraperitoneally with 100 mg/kg gentamicin or neomycin for 4 consecutive days. At the end of the treatment periods, total RNA was extracted from single utricles, reverse transcribed to cDNA and the cDNA amplified by PCR with primers for beta-actin, fimbrin and class III beta-tubulin. Co-amplification allowed quantitative comparison of mRNA between fimbrin, or class III beta-tubulin and beta-actin from the same utricle. Both aminoglycosides, either after 48 h in vitro or immediately after treatment in vivo, caused a significant decrease in the expression of fimbrin mRNA and class III beta-tubulin mRNA, relative to beta-actin mRNA, which itself increased. Light and electron microscopy confirmed that this corresponded to loss of, and damage to, hair cells. The relative expression of fimbrin and class III beta-tubulin mRNAs was partly restored almost to control levels 4 days after cessation of treatment in vivo and fully normalised by 4 weeks, by which time hair cells appeared normal. However, their relative expression remained depressed 4 days after removal of antibiotic in vitro. The iron chelator desferrioxamine (100 microM) in vitro prevented the aminoglycoside-induced reduction in relative expression of mRNA for both fimbrin and class III beta-tubulin. Neither insulin (5 microM) nor a combination of dibutyryl cyclic AMP (5 mM) and the phosphodiesterase inhibitor IBMX (0.5 mM) prevented the decrease in relative expression of the mRNAs for the hair cell-specific proteins, but both treatments allowed their partial recovery in vitro during the 4-day-period after removal of aminoglycoside. It is concluded that the cells of the sensory epithelium of the chick utricle subjected to aminoglycoside-induced damage undergo a process in which mRNA expression is switched away from the production of functional proteins and towards proteins necessary for structural re-organisation. The restoration of mRNA expression to a normal pattern is promoted by the growth factor insulin and by cyclic AMP.

Axonal transport of protein in motor fibres of experimentally diabetic rats--fast anterograde transport.

Fast axonal transport of radiolabelled proteins in motor fibres of rat sciatic nerves was studied after 14 days of streptozotocin-induced diabetes. The rate of fast transport as measured at two time intervals after application of [3H]leucine to the motor neurone cell bodies in the spinal cord was reduced by 21% in diabetic rats. There was no significant change in the time between injection of isotope and the start of fast transport. The amount of axonal transport of radiolabelled proteins as measured by accumulation of proteins proximal to a ligation on the sciatic nerve was also unchanged. The reduction in fast transport rate in the diabetic rats was eliminated by maintenance of normal blood glucose levels in twice daily insulin administration. The results are discussed with regard to the known effects of experimental diabetes on axonal transport in sensory fibres and to the role of fast axonal transport in peripheral neuropathies in general.

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.

Slow axonal transport or proteins; blockade by interruption of contact between cell body and axon.

The influence of ligation and colchicine treatment on the axonal transport of slowly migrating [3H]leucine-labelled proteins was studied in the vagus nerve of the rabbit. Two days after [3H]leucine labelling of the dorsal motor nucleus of the vagus nerve, ligation or local application of 60 mM colchicine immediately blocked the further progression of slowly migrating proteins distal to the site of treatment. Application of 50-100 mug colchicine to the nerve cell bodies 2 days after labelling blocked the transport of slowly migrating proteins within the next 24 h. It is suggested that contact between nerve cell body and the axon is necessary for the maintenance of the slow transport of proteins in these nerves.

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.

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.

Changes in fast axonally transported proteins in the regenerating rabbit vagus nerve.

Regeneration was induced in rabbit vagus nerves by a crush injury. Fourteen and 50 days later [35S]methionine-radiolabelled, fast axonally transported proteins were separated by one- and two-dimensional electrophoresis. Quantitative densitometric analysis of fluorographs from one-dimensional separations showed increases in the radiolabelling of fast transported proteins of 45 and 20 kDa, and a decrease in the radiolabelling of a 26 kDa protein 14 days after crush injury, which were confirmed by two-dimensional separations. These increases were maintained at 50 days post-crush. The proteins have similar molecular weights and isoelectric points to two growth-associated proteins known to have important roles in growth and regeneration.

Tricresyl phosphate inhibits the formation of axon-like processes and disrupts neurofilaments in cultured mouse N2a and rat PC12 cells.

Tricresyl phosphate (1 microg/ml) inhibited the outgrowth of axon-like processes in mouse N2a neuroblastoma and rat PC12 pheochromocytoma cell lines induced to differentiate by serum withdrawal and nerve growth factor addition, respectively. By contrast, it had no effect on the outgrowth of processes by rat C6 glioma cells induced to differentiate with sodium butyrate. The effect on axon outgrowth in the two neuronal cell lines correlated with altered distribution of neurofilament proteins, as determined by indirect immunofluorescence with monoclonal antibody RMd09. Western blots of neuronal cell extracts probed with the same antibody revealed decreased cross-reactivity after exposure to tricresyl phosphate. The results suggest that tricresyl phosphate has a selective effect on neuronal cell differentiation, which involves impaired axon outgrowth, reduced levels of the neurofilament heavy chain and disruption of the neurofilament network.

Limitations to the usefulness of N-succinimidyl propionate for the study of retrograde axonal transport.

Retrograde axonal transport of radiolabelled proteins was studied in rat sciatic nerve, after direct application of [3H]N-succinimidyl propionate. Waves of radiolabelled proteins were observed but only two proteins were predominantly labelled, one of molecular weight 68 kilodaltons (K) and the other of 19K. There was no evidence to confirm the waves as representing retrograde axonal transport of identifiable proteins, and the tendency of the covalent label to bind selectively in vivo to a small number of prominent proteins limits its usefulness for the detection of retrogradely transported proteins in general.