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Biomedical subjects

M Wallin

Publications and source records attributed to M Wallin.

At least 73 records · Page 4Linked to original sources
PubMed1991

A calmodulin inhibitor with high specificity, compound 48/80, inhibits axonal transport in frog nerves without disruption of axonal microtubules.

The calmodulin inhibitor compound 48/80 has previously been shown to arrest axonal transport in vitro in the regenerating frog sciatic nerve. The inhibition was limited to the outgrowth region of nerves, which had been allowed to regenerate in vivo for 6 days after a crush lesion, before they were incubated with or without drugs in vitro overnight. The effects of compound 48/80 on the regenerating nerve were further investigated. A concentration of compound 48/80 (50 micrograms ml-1), which effectively inhibits axonal transport, did not cause observable changes of the microtubules of regenerating axons in the outgrowth region as judged by electron microscopy. Furthermore, it was shown that also a lower concentration (25 micrograms ml-1) inhibited axonal transport. As a measure of possible metabolic effects, the level of ATP was assessed in the regenerating nerve after exposure to compound 48/80. Compound 48/80 at 25 micrograms ml-1 did not change the level of ATP in the nerve. The assembly of bovine brain microtubule proteins in a cell-free system was unaffected by 25 micrograms ml-1 of compound 48/80 and slightly inhibited by 50 micrograms ml-1. At higher concentrations (greater than 100 micrograms ml-1) assembly of microtubules appeared stimulated, and microtubule spirals as well as closely aligned microtubules could be seen. These effects appeared to be unrelated to the transport effects. The present results indicate that compound 48/80 arrests axonal transport via mechanisms other than destruction of axonal microtubules or interference with the energy metabolism. It is possible that these mechanisms involve inhibition of calmodulin-regulated events essential to the transport.

Adenosine Triphosphate↗
PubMed1990

Proteolytic cleavage of microtubule-associated proteins by retroviral proteinases.

Aspartic proteinases from human immunodeficiency virus type 1 (HIV-1) and avian myeloblastosis virus (AMV) were found to interfere with microtubule assembly. Preincubation of the proteinases with purified brain microtubule proteins (tubulin and microtubule-associated proteins) at low ionic strength (pH 6.8), completely inhibited microtubule assembly. Analysis of microtubule proteins after incubation with proteinase showed no effect on tubulin but extensive cleavage of the microtubule-associated proteins 1 and 2 was observed. The digestion by the two proteinases differed. In the presence of HIV-1 proteinase, a fragment with an Mr of approximately 300, appeared, as well as at least three other new fragments, with Mr values of 188,000, 124,000 and 73,000. In the presence of AMV proteinase, the microtubule-associated proteins were extensively digested to many small fragments. The extending microtubule-associated proteins normally seen by electron microscopy on the microtubule surface disappeared after treatment with AMV proteinase. Our results show that retroviral proteinases are not restricted to cleavage of viral polyproteins in vitro. It is suggested that proteolysis of microtubular proteins by viral proteinases is an important step in viral pathogenicity and that it may be part of a mechanism causing degenerative effects in infected cells.

Animals↗
PubMed1989

Correlation between plasma levels of growth factors and von Willebrand factor.

Intimal proliferation of smooth muscle cells is an important characteristic of developing atherosclerotic lesions and late occlusion of venous bypass grafts. Platelet-derived growth factor, released from aggregating platelets at sites of endothelial injury, has been suggested as a main factor responsible for intimal hyperplasia. The von Willebrand factor is a platelet binding protein secreted by endothelial cells and increased plasma levels of this factor has been identified as a marker of endothelial injury. In the present study we have analysed plasma levels of von Willebrand factor and growth factors in healthy controls, young post-infarction patients and patients with recent coronary bypass surgery. The results demonstrate a significant correlation between plasma growth factor activity and the level of von Willebrand factor (R = 0.52, p less than 0.01) and support the notion of a coupling between endothelial injury and release of platelets mitogens.

Adult↗
PubMed1989

Proteolytic cleavage of high-molecular-weight microtubule-associated proteins by the prostatic estramustine-binding protein.

The rat prostatic estramustine-binding protein was found to inhibit assembly of microtubules in a concentration-dependent manner. The inhibition was caused by a proteolytic cleavage of the high-molecular-weight microtubule associated proteins (MAPs), as judged by sodium dodecyl sulfate-gel electrophoresis. A proteolytic fragment with a molecular weight of 199 kDa appeared, which remained bound to the assembled microtubules. Fragments of lower molecular weights (170, 149 kDa) were also found, but they did not bind to the assembled microtubules. Fragments with identical molecular weights were also found after incubation of purified MAP2 with the estramustine-binding protein, indicating that the fragments derive from MAP2. No proteolysis of tubulin, albumin, or casein was found. The estramustine-binding protein was found to be a Zn2+-dependent protease; it was inhibited by EDTA and reactivated by addition of 1 mM Zn2+. Its proteolytic activity was not affected by binding of the antimitotic drug estramustine.

Animals↗
PubMed1988

Effects of proteolysis of the extending parts of the high-molecular-weight microtubule-associated proteins on interactions between microtubules.

Digestion of assembled microtubules with agarose-bound trypsin was performed to obtain microtubules which lack the extending projections, the non-tubulin-binding part of the high-molecular-weight microtubule-associated proteins. The assembly kinetics and the minimum protein concentration for assembly were the same for these trypsinated microtubules as for normal, untreated microtubules. Furthermore, the digested microtubules gave rise to the same change in turbidity per polymer mass as that found for normal microtubules. However, electron microscopy of pelleted microtubules revealed a closer packing after trypsin treatment. A substantially lower increase in specific viscosity was found upon assembly. At concentrations of above approx. 1.5 mg/ml, the viscosity of trypsin-treated microtubules was almost independent of the protein concentration, in contrast to the turbidity, which still increased. Both microtubules and the trypsin-digested microtubules were easily oriented by shear, although the flow linear dichroism signal for the microtubules after trypsin treatment was only half of that found for perfectly oriented normal microtubules. At higher shear force gradients, digested microtubules aggregated side by side as shown by electron microscopy. This was not found for normal microtubules. Even although the extending parts of the high-molecular-weight proteins are not needed for assembly, they were found to play an important role in microtubule orientation and interactions between microtubules, probably by acting as spacers between microtubules.

Animals↗
PubMed1988

Proteolysis of tubulin and microtubule-associated proteins 1 and 2 by calpain I and II. Difference in sensitivity of assembled and disassembled microtubules.

Calpain I and II (EC 3.4.22.17) are Ca2+-activated neutral thiol-proteases. Isolated brain tubulin and microtubule-associated proteins were found to be good substrates for proteolytic degradation by brain calpain I and II. The assembly of microtubules was totally inhibited when the calpains were allowed to act on microtubule proteins initially, and a complete disassembly was found after addition of calpain I to assembled microtubules. The high-molecular weight microtubule-associated proteins were degraded within a few minutes following incubation with calpain as shown by SDS-polyacrylamide gel electrophoresis and electron microscopy. When calpain was added to pre-formed microtubules, either in the presence or in the absence of microtubule-associated proteins, the proteolysis was significantly reduced. When tubulin was pre-assembled by taxol, the formation of proteolytic fragments was decreased indicating that assembly alters the availability of tubulin sites for proteolytic cleavage by calpain. Digested tubulin spontaneously formed aberrant polymers. No considerable change of apparent net charge was seen, thus indicating that calpain cleaves off fragments containing neutral amino acid residues and/or that the fragments of tubulin remain associated as an entity with the same charge as native tubulin. The results suggest that the calpains act as irreversible microtubule regulators.

Animals↗
PubMed1987

Effect of estramustine phosphate on the assembly of trypsin-treated microtubules and microtubules reconstituted from purified tubulin with either tau, MAP2, or the tubulin-binding fragment of MAP2.

Estramustine phosphate, an estradiol nitrogen-mustard derivative is a microtubule-associated protein (MAP)-binding microtubule inhibitor, used in the therapy of prostatic carcinoma. It was found to inhibit assembly and to induce disassembly of microtubules reconstituted from phosphocellulose-purified tubulin with either tau, microtubule-associated protein 2, or chymotrypsin-digested microtubule-associated protein 2. Estramustine phosphate also inhibited assembly of trypsin-treated microtubules, completely depleted of high-molecular-weight microtubule-associated proteins, but with their microtubule-binding fragment present. In all cases estramustine phosphate induced disassembly to about 50%, at a concentration of approximately 100 microM, at similar protein concentrations. However, estramustine phosphate did not affect dimethyl sulfoxide-induced assembly of phosphocellulose-purified tubulin. Estramustine phosphate is a reversible inhibitor, as the nonionic detergent Triton X-100 was found to counteract the inhibition in a concentration-dependent manner. The reversibility was nondisruptive, as Triton X-100 itself did not affect microtubule assembly, microtubule protein composition, or morphology. This new reversible MAPs-dependent inhibitor estramustine phosphate affects the tubulin assembly, induced by tau, as well as by the small tubulin-binding part of MAP2 with the same concentration dependency. This indicates that tau and the tubulin-binding part of MAP2, in addition to their assembly promoting functions also have binding site(s) for estramustine phosphate in common.

Binding Sites↗
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

Changes in the hydrodynamic properties of microtubules induced by taxol.

Microtubule assembly was followed and monitored by (1) the turbidity at 350 nm, (2) the weight of the pelleted microtubules, (3) linear dichroism, LD tau, of the turbidity upon flow orientation, (4) the specific viscosity, eta spec, and (5) electron microscopy. These five methods showed the same features for normal microtubule assembly, but were different in the presence of taxol, a drug which binds to tubulin. The The apparent steady state of microtubule assembly in the presence of taxol as found by turbidity or the weight of pelleted polymer did not represent a stable state, as both LD tau and eta spec continued to change for a much longer time. Microtubules assembled in the presence of taxol from microtubule proteins as well as from purified tubulin were difficult to orient, as high flow gradients were needed and the maximal LD tau value represented only 20% of the LD tau for normal microtubules. In contrast to the slow relaxation of normal microtubules, rapid relaxation to random orientation was found in the presence of taxol. Low orientability was also indicated by electron micrographs, in which pelleted microtubules were seen to be randomly oriented in the presence of taxol. Taxol induced a very high eta spec, 4-times the steady-state value in the initial phase of assembly, which slowly declined again to a steady state, an effect which was also found for assembly of purified tubulin assembled in the absence of the microtubule-associated proteins. The presence of taxol did not change the relative amount and composition of the microtubule-associated proteins in the assembled microtubules. The results therefore suggest that taxol alters the hydrodynamic properties of the microtubules due to its interaction with tubulin and that this alteration is not an effect of the microtubule-associated proteins.

Alkaloids↗
PubMed1985

The binding of Ruthenium red to tubulin.

The inhibition of microtubule assembly by Ruthenium red (Deinum, J., Wallin, M., Kanje, M. and Lagercrantz, C. (1981) Biochim. Biophys. Acta 675, 209-213) could be counteracted by either taxol or dimethyl sulfoxide. Ruthenium red remained bound to the assembled microtubules. Microtubules assembled in the presence of Ruthenium red and taxol showed the typical taxol-dependent stability. The dimethyl sulfoxide-induced microtubules showed normal assembly characteristics, e.g., were GTP dependent, could be disassembled by cold, colchicine and Ca2+ and had no alterations in ultrastructure. The absolute disassembly induced by Ca2+ in the presence of dimethyl sulfoxide and Ruthenium red was dependent on the microtubule protein concentration, but independent in the absence of Ruthenium red. Ruthenium red was strongly bound to purified tubulin also in the presence of 8% (v/v) dimethyl sulfoxide. The dimethyl sulfoxide-induced assembly of purified tubulin in the presence of Ruthenium red was slightly stimulated, although the critical protein concentration was the same. It was found by resonance Raman spectroscopy with a flow technique that Ruthenium red did not bind to a specific calcium binding site on tubulin, although binding to a GTP binding site cannot be excluded. The wavenumbers of the lines in the region 375-500 cm-1 differ from those found for Ruthenium red bound to typical calcium-binding proteins such as calmodulin. Although Ruthenium red binds to serum albumin as well, the spectrum with albumin resembled that of the free dye.

Alkaloids↗
PubMed1985

Interaction of estramustine phosphate with microtubule-associated proteins.

We have reported [(1984) Cancer Res., in press] that estramustine phosphate inhibits microtubule assembly and disassembled preformed microtubules. We now present evidence that estramustine phosphate inhibits microtubule assembly by binding to the microtubule-associated proteins. We have found that: additional microtubule-associated proteins relieved the inhibition of assembly by estramustine phosphate; 3H-labelled estramustine phosphate bound predominantly to the microtubule-associated proteins; and the content of the microtubule-associated proteins was reduced in taxol reversed estramustine phosphate-inhibited microtubules.

Alkaloids↗
PubMed1985

Diethylstilbestrol induces metaphase arrest and inhibits microtubule assembly.

Diethylstilbestrol produced a dose-dependent increase in the mitotic index of the human prostatic tumour cell line DU 145. This is the result of metaphase arrest which may be induced by the action of diethylstilbestrol on spindle microtubules. Evidence is presented to show that diethylstilbestrol affects microtubules. Diethylstilbestrol completely inhibited the assembly of isolated brain microtubules although only partial disassembly could be induced. In contrast to other microtubule poisons, the inhibitory effect of diethylstilbestrol on taxol-induced self-assembly could be reversed by the addition of GTP.

Alkaloids↗