Biomedical subjects

M Kanje

Publications and source records attributed to M Kanje.

At least 91 records · Page 5Linked to original sources

PubMed1987

Regeneration of the sciatic nerve in mice and rats exposed to trichloroethylene.

The effects of trichloroethylene (TCE) on regeneration of the sciatic nerve after a crush lesion was tested in mice and rats. A lesion was made on nerves in unexposed animals and in animals pre-exposed to TCE for 20 days. Experimental animals were then exposed to TCE for an additional 4 days. Exposures were continuous at 150 or 300 ppm. Regeneration was measured by pinching the outgrowing nerve fibers. Regeneration was retarded in the TCE-exposed animals compared to the air-exposed controls. Mice were more sensitive to TCE exposure than rats. Liver weight increased in TCE-exposed mice but there was no correlation between this effect and that on nerve regeneration.

Administration, Inhalation↗

PubMed1987

Calmodulin-binding proteins within the slow phase of axonal transport in the rabbit vagus nerve.

Calmodulin-binding proteins (CBPs) in the rabbit vagus nerve were studied by means of calmodulin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis. The soluble fraction (10(5) g supernatant) of a nerve homogenate contained four CBPs with molecular weights of 44, 55, 91, and 93 kD, respectively. Slowly transported proteins were recovered in the vagus 3 days after injection of [35S]methionine into the nodose ganglion. Four labelled CBPs with molecular weights of 44, 55, 69, and 83 kD, respectively were found. The nodose ganglion contained two labelled CBPs, 44 and 55 kD. The 55-kD CBP was identified as tubulin after immunoblotting. In separate experiments it was also shown that bovine brain tubulin bound to the calmodulin column.

Animals↗

PubMed1986

Ornithine decarboxylase activity in dorsal root ganglia of regenerating frog sciatic nerve.

Ornithine decarboxylase (ODC) activity was studied in dorsal root ganglia (DRG) of regenerating frog sciatic nerve. There was a significant increase in activity two days after a crush lesion of the nerve 2.5 cm distal to the DRG. The increase reached a maximum after 7 days, then declined but remained above control levels for at least 9 days. An endoneural injection of vinblastine, a potent inhibitor of retrograde and orthograde axonal transport, between the DRG and the crush inhibited the increase in ODC. In contrast, injections of vinblastine into undamaged nerves failed to affect ODC. The increase in ODC and also the regenerative properties of the nerve could be prevented by daily i.p. injections of alpha-difluoromethyl ornithine. We suggest that a signal is formed at the site of injury in the sciatic nerve. This signal is conveyed to the DRG by retrograde axonal transport where it initiates the events leading to an increase in ODC. This increase appears to be necessary for the regeneration of sensory fibers in the frog sciatic nerve.

Animals↗

PubMed1986

A calcium-activated neutral protease in the frog nervous system which degrades rapidly transported axonal proteins.

A Ca2+-activated protease (CANP) was prepared from a soluble extract of the frog nervous system and separated from an endogenous high-molecular weight inhibitor by ion exchange chromatography. Its proteolytic action was tested on rapidly transported [3H]leucine-labelled proteins from sensory axons of the sciatic nerve. The enzyme was activated at neutral pH by Ca2+ at millimolar level, and was inhibited by leupeptin and the endogenous inhibitor. Its most pronounced degradative action was exerted towards two prominent rapidly transported peaks corresponding to 82 kDa and 90 kDa proteins. Proteolysis was accompanied by the appearance of mainly 16 kDa degradation products. When newly synthesized soluble proteins of the spinal dorsal ganglia were used as substrates, the presence of CANP resulted in degradation of proteins in a broad molecular-weight range (40-200 kDa).

Animals↗

PubMed1986

The effect of gossypol on fast axonal transport and microtubule assembly.

Gossypol at micromolar concentrations (2 microM) was found to inhibit axonal transport and a microsomal ATPase activity in the frog sciatic nerve, although axonal microtubules and the neuronal content of AMP, ADP and ATP were not affected. At slightly higher concentrations (30-40 microM), gossypol also inhibited microtubule assembly and neuronal energy metabolism. Gossypol accumulated in the nerve and the results indicate that gossypol may act as a potent neurotoxin.

Adenosine Triphosphatases↗

PubMed1986

Polyamine depletion increases cellular ribonucleotide levels.

Depletion of the putrescine and spermidine content of Ehrlich ascites tumor cells by alpha-difluoromethylornithine (DFMO) treatment results in at least a 1 500-fold increase in the decarboxylated S-adenosylmethionine (deSAM) content. The accumulation of this adenine nucleoside occurs because of the absence of putrescine and spermidine to act as aminopropyl group acceptors in the spermidine and spermine synthase reactions and because of an increase in S-adenosylmethionine decarboxylase activity. The fact that the synthesis of deSAM continues in DFMO-treated cells makes the pathway an adenine trap. This prompted a study of the adenine nucleotide pools. High-performance liquid chromatographic analysis showed that the total adenine nucleotide pool increased, rather than decreased, as a result of DFMO treatment; the major contributors to the increase being ATP and ADP, which increased 2.6 and 1.9 times, respectively. The cellular content of other ribonucleotides increased as well, particularly that of UTP and CTP. When putrescine was added together with DFMO, the increases in cellular ribonucleotide contents were prevented, showing that they were indeed caused by polyamine depletion.

Adenine Nucleotides↗

PubMed1986

The use of whole-mount preparations of nerves labelled with axonally transported radioactive proteins to study regeneration.

A method for studying the process of regeneration in the frog sciatic nerve is presented. The outgrowth of sensory axons was determined by assaying the distribution of axonally transported radioactive proteins by fluorography of whole-mount nerve preparations. After labelling, the nerves were fixed at their in vivo lengths, impregnated with a fluorographic reagent and whole-mounted on a glass-slide before exposure to an X-ray film. In this way the rate of axon elongation after nerve crush lesions could be accurately determined. Nerve regeneration preceded by a conditioning lesion will be described. After the outgrowth has been measured the method enables subsequent analysis of, for instance, the protein composition in selected parts of the same nerve. The technique should be broadly applicable for studying nerve regeneration.

Animals↗

PubMed1985

Ca2+-activated protease activity in frog sciatic nerve: characterization and effect on rapidly transported axonal proteins.

Protease activity was studied in the frog sciatic nerve. The activity was measured as the release of TCA-soluble radioactivity from either 3H-labelled proteins transported by rapid axonal transport (AXT) or 3H-labelled ganglionic proteins. In nerve homogenates containing transported substrates, protease activity exhibited two peaks, one around pH 5 and one around pH 8. Ca2+ at 100 microM or higher concentrations only stimulated the latter, which was inhibited by 1 mM parachloromercuric benzoate, a sulphydryl reagent, but unaffected by ATP (1 mM). The proteolytic activity was recovered in the 10(5) g supernatant of the homogenate. In desheathed nerves containing 3H-labelled transported proteins, the protease activity could be activated by exposing the nerve to a Ca2+-ionophore, X-537 A, or to an elevated Ca2+-concentration (50 mM). These conditions were also shown to increase the influx and efflux of 45Ca2+ in the nerves. The results indicate the presence within axons of a Ca2+-activated soluble protease, which degrades rapidly transported proteins. The finding that the protease degraded ganglionic soluble proteins to about the same extent suggests a broad substrate specificity. The present system should be useful for further characterization of protease activity during various physiological conditions.

Adenosine Triphosphate↗

PubMed1985

Effect of estramustine phosphate on the assembly of isolated bovine brain microtubules and fast axonal transport in the frog sciatic nerve.

Estramustine phosphate (0.01 to 0.5 mM), an estradiol mustard derivative used in the therapy of prostatic carcinoma, inhibited the assembly of brain microtubules proteins in vitro and disassembled preformed microtubules. In the presence of estramustine phosphate, the minimum microtubule-protein concentration sufficient for the assembly of microtubules was increased. Low concentrations of taxol (20 microM) completely reversed the inhibition of assembly by estramustine phosphate. The effects were specific to estramustine phosphate since neither estradiol 17 beta-phosphate, the hormonal moiety of the drug, nor nornitrogen mustard, the alkylating moiety, had any effect on assembly. Estramustine phosphate (0.1 to 0.5 mM) was also found to reversibly inhibit fast axonal transport in the frog sciatic nerve. The nerve content of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate was not significantly affected by estramustine phosphate. Our results suggest that the cytotoxic action of estramustine phosphate could be dependent partially on an interaction with microtubules, probably via the microtubule-associated proteins.

Animals↗

PubMed1984

Polyamine biosynthetic enzymes as targets in cancer chemotherapy.

In this chapter we focus attention on recent developments in the biosynthesis of putrescine, spermidine and spermine and their linkage to salvage pathways of methionine and adenine nucleotide synthesis. We describe the use of specific inhibitors of the polyamine biosynthetic enzymes for studying the role of polyamines in cell growth and division as well as in cell differentiation. Some novel findings are presented which suggest that part of the inhibitory action that polyamine synthesis inhibitors exert on DNA synthesis may be due to the accumulation of ADP and ATP. We show that polyamine synthesis inhibitors are capable of inducing terminal differentiation of neoplastic cells to forms with no further proliferative potential, and briefly discuss the potential use of this approach in cancer chemotherapy.

Adenine Nucleotides↗

PubMed1984

Inhibition of fast axonal transport by erythro-9-[3-(2-hydroxynonyl)]adenine.

erythro-9-[3-(2-Hydroxynonyl)]adenine, an inhibitor of protein carboxylmethylation and dynein-ATPase activity, inhibited fast axonal transport in vitro in frog sciatic nerves. Its site of action might be associated with an ATPase on which transport depends, since specific carboxylmethylation inhibitors lacked effects on transport. The levels of high energy phosphates and protein synthesis were unaffected by the drug at a transport-inhibiting concentration, making disturbances due to metabolic effects less likely. An erythro-9-[3-(2-hydroxynonyl)]adenine-sensitive ATPase was looked for in various nerve fractions but has so far not been resolved.

Adenine↗

PubMed1984

Perchloroethylene: effects on body and organ weights and plasma butyrylcholinesterase activity in mice.

The effects of continuous and intermittent inhalation of perchloroethylene (PCE) on plasma butyrylcholinesterase (BuChE) activity, organ weights, liver morphology and motor activity in mice (strain NMRI) were tested. PCE exposure increased plasma BuChE activity in a time- and concentration dependent manner in both sexes. The increase was statistically significant at 37 p.p.m. in animals continuously exposed for 30 days. BuChE increased approximately 1.5 times in females and 2.5 times in males after 120 days exposure to 150 p.p.m. After rehabilitation of animals exposed for 30 days to 150 p.p.m., BuChE levels returned to normal. Liver weight also increased in a time and concentration dependent manner. Both sexes exhibited significant liver enlargement at 9 p.p.m. The increase was about 2.3 in females and 1.9 in males after continuous exposure to 150 p.p.m. for 120 days. After rehabilitation (120 days) of animals exposed to 150 p.p.m. for 30 days, a 10% increase still remained. A decrease in body weight gain was seen in both sexes after exposure to concentrations above 75 p.p.m. Female kidney weight was slightly increased. No clear effect on spleen weight could be detected. When the same time-weighted average concentration was used, intermittent exposure for 30 days had similar effects on liver weight and BuChE activity as continuous exposure, even when exposures lasted for only one hour per day. Liver cell morphology was changed after PCE exposure. The alterations could be observed already at 9 p.p.m. but disappeared after rehabilitation.

Animals↗

PubMed1984

Is protease activity involved in fast axonal transport?

N-alpha-p-Tosyl-L-Lysine Chloromethyl Ketone (TLCK), a protease inhibitor, was found to irreversibly inhibit rapid axonal transport of protein in vitro in the frog sciatic nerve. TLCK exerted its action at the axonal level and seemed to depress the rate rather than the amount of transported protein. The efficiency of TLCK as a protease inhibitor was demonstrated by polyacrylamide gel electrophoresis, which showed that degradation of high molecular weight proteins (presumably neurofilament subunits) into a 25000 dalton protein could be induced by exposing the frog nerves to triton-X and prevented by the presence of TLCK. Findings that TLCK, at a transport inhibiting concentration (0.1 mM), had little or no effects on either protein synthesis or ATP levels, suggest that TLCK did not affect transport due to general cytotoxic properties. The effects of TLCK is discussed in relation to possible roles of protease activity in axonal transport.

Adenosine Triphosphate↗

PubMed1983

Trichloroethylene: further studies of the effects on body and organ weights and plasma butyrylcholinesterase activity in mice.

The effects of continuous and intermittent inhalation of trichloroethylene (TCE) were studied in male and female mice. Plasma butyrylcholinesterase (BuChE) activity, body, liver, kidney and spleen weights were measured. The liver was studied histologically and motor activity measured with doppler radar. Continuous TCE-exposure (37-300 p.p.m.) increased plasma BuChE activity in the males in a time and concentration dependent manner. After 30 days at 37 p.p.m. the increase was about 25%. Exposure to 300 p.p.m. for 30 days increased the activity three times. BuChE activity in females was only slightly influenced even at 300 p.p.m. Liver weight was increased in a time and concentration dependent manner in both sexes. In animals continuously exposed for 30 days to 300 p.p.m., liver weight was roughly twice that of the air-exposed controls. Morphological changes were observed in the liver of TCE-exposed animals. Above 150 p.p.m. kidney weight in both sexes was significantly increased. This effect was more pronounced in the males than in the females. Spleen weight was not influenced by the exposure. Body weight increase was slightly lower in exposed animals. Plasma BuChE activity and liver weight returned to normal when exposure was terminated. Intermittent exposure to short pulses of high concentration of TCE had roughly the same effect on BuChE, body and organ weights as continuous exposure to the same time-weighted average. Motor activity was affected by the intermittent exposure schedules. At 900 p.p.m. decrease in activity was observed. At 3600 p.p.m. motor activity was considerably increased.

Animals↗

PubMed1982

Divalent cations and fast axonal transport in chemically desheathed (Triton X-treated) frog sciatic nerve.

We have studied the ability of divalent cations to restore to normal axonal transport (AXT) which was inhibited by deprivation of Ca2+ and/or Mg2+ ions. The epi- and perineurium of the frog sciatic nerve were damaged by a 30-s wash in Triton X-100 containing frog Ringer's. This treatment did not affect either AXT or nerve levels of Ca2+ and Mg2+, but made the ions more easily extractable with a Ca2+- and Mg2+-free Ringer's solution (CMFR). Inhibition of AXT was achieved by incubating Triton X-100-treated nerves in CMFR + EGTA for 5 h, followed by an additional incubation for 12 h in CMFR or Ringer's devoid of only Ca2+ (CFR). These treatments reduced Ca2+ and Mg2+ contents by 77% and 38% respectively. Addition of Ca2+ (1.1 mM) during the 12-h period stimulated AXT, measured as accumulation of 3H-labelled components in front of a ligature, several fold. Mg2+ could not substitute for Ca2+ but potentiated the stimulating effect of Ca2+. Addition of other divalent cations did not affect AXT (Sr2+ and Ba2+) or potentiated the inhibition caused by Ca2+-deprived medium (Mn2+ and Co2+). ATP and creatine phosphate contents were similar in nerves incubated in Ca2+-deprived medium and in Ca2+-containing Ringer's. Thus, inhibition of AXT in the former situation was not due to a decreased availability of high energy phosphates. Two calcium antagonists, D-600 and nifedipin, which are potent smooth muscle relaxants, effectively blocked AXT. The present results suggest that Ca2+ is specifically required to maintain AXT and that an analogy exists between Ca2+ regulation during smooth muscle contraction and AXT.

Animals↗

PubMed1982

Effects of phenothiazines and dibenzazepines on axonal transport and microtubule assembly in vitro.

Various phenothiazines (thioridazine, trifluoperazine and chlorpromazine) and dibenzazepines (lofepramine, amitriptyline and desipramine) were studied for effects on fast axonal transport (AXT) in vitro in frog sciatic nerves. AXT, measured as the accumulation of (3H) leucine-labelled proteins in front of a ligature, was inhibited by more then 50% by all the drugs tested at 0.2 mM concentrations. Thioridazine and lofepramine were the most potent inhibitors. These effects were not due to decreased ganglionic incorporation. Some of the drugs also reduced the levels of high energy phosphates, adenosinetriphosphate (ATP) and creatinephosphate (CrP), but not to an extent which is likely to explain the arrested AXT. The polymerization of purified brain tubulin was inhibited by the phenothiazines but unaffected by the dibenzazepines at concentrations which inhibited AXT. Phenothiazines and dibenzazepines are chemically related and known to have a high affinity for calmodulin. The possibility that these drugs interefere with calmodulin regulated processes of importance for AXT will be discussed.

Adenosine Triphosphate↗

PubMed1981

Inhibition of rapid axonal transport in vitro by the ionophores X-537 A and A 23187.

The effects of the ionophores X-537 A and A 23187 on axonal transport (AXT)' high energy phosphates, levels of Ca2+ and ultrastructure were investigated in frog sciatic nerves in vitro. X-537 A at 0.09 muM blocked AXT of [3H]labelled proteins by 50% as judged by ligature experiments while the levels of ATP and CrP remained unchanged. Pulse-label experiments showed that the amount of transported material decreased whereas the rate of AXT was only slightly retarded. Elevated extracellular levels of Ca2+ (5mM) potentiated the inhibitory effect of the ionophore. In contrast, ruthenium red counteracted the ionophore induced inhibition of AXT. A 23187 at 10 muM but not at 2 muM inhibition AXT but also reduced the ATP and CrP levels. In pulse-label experiments A 23187 displayed similar but less pronounced effects than X-537 A. Both ionophores increased the total calcium content of the nerve and appeared to decrease the amount of axonal mictotubles (MT). As X-537 is a very potent inhibitor of AXT it may prove a valuable tool in exploring the relations between AXT, Ca2+ and MT.

Adenosine Triphosphate↗

PubMed1981

Effects of neurotransmitters on calcium efflux from cultured glioma cells.

The effects of various neurotransmitters and cyclic nucleotides on 45Ca2+ efflux in cultured human glioma cells were investigated. Glutamate and glycine, but not GABA, stimulated 45Ca2+ release from the cells. Stimulation of beta-adrenergic receptors but not alpha-adrenergic receptors also increased 45Ca2+ efflux. Cholinergic receptor stimulation by carbachol had the same effect. The stimulatory effect of carbachol was abolished in the presence of either atropine or hexamethonium. C-AMP and c-GMP increased the 45Ca2+ efflux, suggesting that these agents are involved in the transmitter-stimulated release of 45Ca2+ from the cell. Kinetic analysis of the efflux revealed four calcium compartments. The carbachol-stimulated efflux represented a net release of calcium and could be ascribed to the slowest compartment. The physiological role of the transmitter-stimulated calcium release is discussed in terms of calcium-regulated stimulus-response coupling in glial-neural interaction during excitation.

Astrocytoma↗