Magnetic Resonance Image-Guided Radiotherapy (MRIgRT): A 4.5-Year Clinical Experience.

AIMS: Magnetic resonance image-guided radiotherapy (MRIgRT) has been clinically implemented since 2014. This technology offers improved soft-tissue visualisation, daily imaging, and intra-fraction real-time imaging without added radiation exposure, and the opportunity for adaptive radiotherapy (ART) to adjust for anatomical changes. Here we share the longest single-institution experience with MRIgRT, focusing on trends and changes in use over the past 4.5 years. MATERIALS AND METHODS: We analysed clinical information, including patient demographics, treatment dates, disease sites, dose/fractionation, and clinical trial enrolment for all patients treated at our institution using MRIgRT on a commercially available, integrated 0.35 T MRI, tri-cobalt-60 device from 2014 to 2018. For each patient, factors including disease site, clinical rationale for MRIgRT use, use of ART, and proportion of fractions adapted were summated and compared between individual years of use (2014-2018) to identify shifts in institutional practice patterns. RESULTS: Six hundred and forty-two patients were treated with 666 unique treatment courses using MRIgRT at our institution between 2014 and 2018. Breast cancer was the most common disease, with use of cine MRI gating being a particularly important indication, followed by abdominal sites, where the need for cine gating and use of ART drove MRIgRT use. One hundred and ninety patients were treated using ART in 1550 fractions, 67.6% (1050) of which were adapted. ART was primarily used in cancers of the abdomen. Over time, breast and gastrointestinal cancers became increasingly dominant for MRIgRT use, hypofractionated treatment courses became more popular, and gastrointestinal cancers became the principal focus of ART. DISCUSSION: MRIgRT is widely applicable within the field of radiation oncology and new clinical uses continue to emerge. At our institution to date, applications such as ART for gastrointestinal cancers and accelerated partial breast irradiation (APBI) for breast cancer have become dominant indications, although this is likely to continue to evolve.

HIPAA compliance questions for business partner agreements.

If your organization is covered by HIPAA, do you know what's expected of you--and of your vendors--with regard to privacy of health information? To make sure your organization is in compliance, contracts with business partners will need careful review. The author offers an overview of the proposed regulations and offers some tips to get started.

HIPAA privacy: "individual rights" and the "minimum necessary" requirements.

This Article focuses on two key aspects of the proposed regulations related to health information privacy published by the Department of Health and Human Services ("DHHS") pursuant to the Health Insurance Portability and Accountability Act of 1996 ("HIPAA"). These two aspects, the provisions regarding individual rights and the provisions requiring use of the minimum amount of information necessary to accomplish a given purpose, will be particularly burdensome for the healthcare industry. Furthermore, they are likely to generate a significant number of complaints to the DHHS Secretary relating to alleged violations of the regulations. This Article analyzes the proposed regulations governing these two issues and offers practical advice regarding steps that entities should take in anticipation of the final regulations.

Identification of betaIII- and betaIV-tubulin isotypes in cold-adapted microtubules from Atlantic cod (Gadus morhua): antibody mapping and cDNA sequencing.

Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30 degrees C. The cold-adaptation is an intrinsic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold-adaptation we have further characterized cod tubulins using alpha- and beta-tubulin site-directed antibodies and antibodies towards posttranslationally modified tubulin. In addition, one cod brain beta-tubulin isotype has been sequenced. In mammals there are five beta-tubulins (betaI, betaII, betaIII, betaIVa and betaIVb) expressed in brain. A cod betaIII-tubulin was identified by its electrophoretic mobility after reduction and carboxymethylation. The betaIII-like tubulin accounted for more than 30% of total brain beta-tubulins, the highest yield yet observed in any animal. This tubulin corresponds most probably with an additional band, designated beta(x), which was found between alpha- and beta-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod beta-tubulin isoforms. The sequenced cod tubulin was identified as a betaIV-tubulin, and a betaIV-isotype was stained by a C-terminal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain beta-tubulin. Based on the estimated amounts of betaIII- and betaIV-tubulins in cod brain, our results indicate that cod has at least one additional beta-tubulin isotype and that beta-tubulin diversity evolved early during fish evolution. The sequenced cod betaIV-tubulin had four unique amino acid substitutions when compared to beta-tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod beta-tubulin. Residues 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin dimer structure to loops that most probably interact with other tubulin molecules within the microtubule, and might contribute to cold-adaptation of microtubules.

Preparation of a monoclonal antibody specific for the class I isotype of beta-tubulin: the beta isotypes of tubulin differ in their cellular distributions within human tissues.

Tubulin, the subunit protein of microtubules, is an alpha/beta heterodimer. In many organisms, both alpha and beta consist of various isotypes. Although the isotypes differ in their tissue distributions, the question of whether the isotypes perform different functions in vivo is unanswered. In mammals, the betaI and betaIV isotypes are quite widespread, and betaII is less so, while betaIII and betaVI have narrow distributions and betaV distribution is unknown. As a tool for localizing the isotypes, we report the preparation of a monoclonal antibody specific for betaI, to add to our previously described monoclonal antibodies specific for betaII, betaIII, and betaIV [Banerjee et al., J. Biol. Chem. 263:3029-3034, 1988; 265:1794-1799, 1990; 267:5625-5630, 1992]. In order to prepare this antibody, we have purified betaI-rich rat thymus tubulin. We have used our battery of antibodies to localize the beta isotypes in four human tissues: oviduct, skin, colon, and pancreas. We have found striking differences in their tissue distributions. There is little or no betaIII in these tissues, except for the columnar epithelial cells of the colon. BetaII is restricted to very few cells, except in the skin, where it is concentrated in the stratum granulosum. BetaI is widespread in all the epithelia. In the skin it is found in the entire stratum malpighii. In the oviduct, betaI is found largely in the nonciliated epithelial cells. In the exocrine pancreas, betaI occurs only in the centroacinar cells and not in the acinar cells; the latter do not stain with any of these antibodies. BetaIV is present at very low levels in skin and pancreas. By contrast, it is prominent in the colon and also in the oviduct, where it occurs in all the epithelial cells, especially in the ciliated cells, with the highest concentrations in the cilia themselves. These results suggest that the regulation of the expression and localization of isotypes in tissues is very complex.

Interaction of bovine brain tubulin with the 4(1H)-pyrizinone derivative IKP104, an antimitotic drug with a complex set of effects on the conformational stability of the tubulin molecule.

The ligands of tubulin have proved to be excellent probes for the conformation of the tubulin molecule. The most varied in their effects on tubulin are those ligands which are competitive or noncompetitive inhibitors of vinblastine binding. The 4(H)-pyrizinone derivative 2-(4-fluorophenyl)-1-(2-chloro- 3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone [sequence: see text] (IKP104) is a novel antimitotic drug which inhibits microtubule assembly in vitro and in vivo and polymerizes tubulin into spiral filaments. Using a fluorescence assay, we found that IKP104 appears to bind to tubulin at two classes of site, differing in affinity. IKP104 also blocks formation of an intrachain cross-link in beta-tubulin, induced by N,N"-ethylenebis(iodoacetamide), linking Cys12 to either Cys201 or Cys211. IKP104 appears to belong to that group of tubulin ligands which includes vinblastine, maytansine, rhizoxin, phomopsin A, dolastatin 10, and halichondrin B. An unusual effect of IKP104 is that it greatly enhances the decay or apparent unfolding or opening of the tubulin molecule. The sulfhydryl titer of tubulin is doubled and the exposure of hydrophobic areas on the tubulin molecule is tripled by IKP104. These effects of IKP104 are counteracted by vinblastine, maytansine, and phomopsin A, suggesting that IKP104 may be competing with these other drugs for binding to tubulin. However, the effects are also counteracted by colchicine and podophyllotoxin, implying a more complex effect, namely, that IKP104 and colchicine, even when both are bound to tubulin, are competing for their effects on the same domain of tubulin. Surprisingly, when IKP104 is used in conjunction with colchicine, binding of colchicine to tubulin is strongly stabilized.(ABSTRACT TRUNCATED AT 250 WORDS)

Common hand warts in athletes: association with trauma to the hand.

Forty-nine members of a university track team and 80 members of the crew team were surveyed about warts on their hands. They were also questioned about the nature and extent of their exercise, the types of equipment they used, and whether they wore protective gloves. Common hand warts were significantly more prevalent in members of the crew team than in members of the track team (25% v 10%; p < .05). Although both groups lifted weights regularly, the crew team members were less likely to use protective gloves; they sustained additional trauma to their hands from almost daily exercise on rowing machines and river practice. College health providers should question patients with hand warts about types of athletic activity and should suggest that they protect their hands by wearing weight-lifter's gloves.

Interaction of ustiloxin A with bovine brain tubulin.

Ustiloxin A is a modified peptide derived from false smut balls on rice panicles, caused by the fungus Ustilaginoidea virens; structurally, it resembles phomopsin A. Ustiloxin A is cytotoxic and is an inhibitor of microtubule assembly in vitro. Because of its resemblance to phomopsin A, we examined its interaction with tubulin and compared the results with those obtained with phomopsin A and dolastatin 10, both of which were found previously to have very similar effects. We determined that ustiloxin A inhibited the formation of a particular intra-chain cross-link in beta-tubulin, as do vinblastine, maytansine, rhizoxin, phomopsin A, dolastatin 10, halichondrin B and homohalichondrin B; this is in contrast to colchicine and podophyllotoxin which do not inhibit formation of this cross-link. Ustiloxin A also inhibited the alkylation of tubulin by iodo[14C]acetamide, as do phomopsin A and dolastatin 10; vinblastine was almost as potent as inhibitor of alkylation as ustiloxin A, whereas maytansine, halichondrin B and homohalichondrin B have little or no effect. In addition, ustiloxin A inhibited exposure of hydrophobic areas on the surface of the tubulin molecule. In this respect, ustiloxin A was indistinguishable from phomopsin A but slightly more effective than dolastatin 10 and considerably more effective than vinblastine; this provides a strong contrast to maytansine, rhizoxin, and homohalichondrin B which have no effect on exposure of hydrophobic areas and to halichondrin B which enhances exposure. Lastly, ustiloxin A strongly stabilized the binding of [3H]colchicine to tubulin. The combination of ustiloxin A with cholchicine stabilized tubulin with a half-life of over 8 days, comparable with results obtained with phomopsin A and colchicine. A comparison of the structures of ustiloxin A, phomopsin A and dolastatin 10 raised the possibility that the strong stabilization of the tubulin structure may require a short segment of hydrophobic amino acids such as the modified valine-isoleucine sequence present in all three compounds. The rest of the structure, specifically the large ring of ustiloxin A and phomopsin A, may serve to place this sequence in an appropriate conformation to interact with tubulin.

Interaction of ethacrynic acid with bovine brain tubulin.

Ethacrynic acid is a diuretic agent that reacts with sulfhydryl groups in proteins, and which shows promise of effectiveness in the treatment of glaucoma. Ethacrynic acid is a known inhibitor of microtubule assembly in vitro (Xu et al., Arch Biochem Biophys 296: 462-67, 1992). We have used N,N'-ethylenebis (iodoacetamide) (EBI) as a probe to examine the sulfhydryl groups of tubulin; EBI can form two intra-chain cross-links in beta-tubulin. One of these, beta*, connects Cys239 with Cys354; the other, beta s, joins Cys12 with either Cys201 or Cys211 (Little and Ludueña, EMBO J 4: 51-56, 1985; Biochim Biophys Acta 912: 28-33, 1987). Formation of beta * inhibits microtubule assembly in vitro, consistent with the hypothesis that Cys239 has an assembly-critical sulfhydryl (Bai et al., Biochemistry 28: 5606-5612, 1989). We have examined the interaction of ethacrynic acid with the sulfhydryl groups of bovine brain tubulin. We found that 130 microM ethacrynic acid gave half-maximal inhibition of assembly, but had no effect on the formation of the beta * cross-link by EBI. Ethacrynic acid, however, did inhibit substantially formation of the beta s cross-link at this concentration and half-maximally inhibited it at approximately 185 microM. Half-maximal inhibition of the alkylation of tubulin sulfhydryls by iodo [14C]acetamide was obtained at an ethacrynic acid concentration in the range of 190-325 microM. These results indicate that ethacrynic acid can inhibit microtubule assembly by reacting with sulfhydryl groups other than those of Cys239 and Cys354 and suggest that other sulfhydryl groups in tubulin could be assembly-critical. These results also raise the possibility that these other assembly-critical sulfhydryls may be those of Cys12, Cys201 or Cys211.

Differential effects of active isomers, segments, and analogs of dolastatin 10 on ligand interactions with tubulin. Correlation with cytotoxicity.

Dolastatin 10 is a potent antimitotic peptide isolated from the marine mollusk Dolabella auricularia. Four of its five residues are modified amino acids (in sequence, dolavaline, valine, dolaisoleuine, dolaproine, dolaphenine). Besides inhibiting tubulin polymerization, dolastatin 10 non-competitively inhibits vinca alkaloid binding to tubulin, inhibits nucleotide exchange and formation of the beta s cross-link, and stabilizes the colchicine binding activity of tubulin. To examine the mechanism of action of dolastatin 10 we prepared six chiral isomers, one tri- and one tetrapeptide segment, and one pentapeptide analog of dolastatin 10, all of which differ little from dolastatin 10 as inhibitors of tubulin polymerization. However, only two of the chiral isomers were similar to dolastatin 10 in their cytotoxicity for L1210 murine leukemia cells and in their effects on vinblastine binding, nucleotide exchange, beta s cross-link formation, and colchicine binding. These were isomer 2, with reversal of configuration at position C(19a) in the dolaisoleuine moiety, and isomer 19, with reversal of configuration at position C(6) in the dolaphenine moiety. The pentapeptides with reduced cytotoxicity and reduced effects on tubulin interactions with other ligands were all modified in the dolaproine moiety at positions C(9) and/or C(10). The tripeptide and tetrapeptide segments which inhibited polymerization but not ligand interactions were the amino terminal tripeptide (lacking dolaproine and dolaphenine) and the carboxyl terminal tetrapeptide (lacking dolavaline). We speculate that strong inhibition of other ligand interactions with tubulin requires stable peptide binding to tubulin (i.e. slow dissociation), but that inhibition of polymerization requires only rapid binding to tubulin.

Interaction of halichondrin B and homohalichondrin B with bovine brain tubulin.

Halichondrin B is a polyether macrolide of marine origin which binds to tubulin and inhibits microtubule assembly in vitro and in vivo. As is the case with phomopsin A and dolastatin 10, halichondrin B is a non-competitive inhibitor of vinblastine binding to tubulin. Analogous to maytansine, which by contrast is a competitive inhibitor of vinblastine binding, halichondrin B has no effect on colchicine binding, which is greatly stabilized by phomopsin A and dolastatin 10, but not by maytansine. We have previously developed assays which allow sensitive discrimination among the interactions of various ligands with tubulin, and examined the effects of ligands on the reactivity of tubulin sulfhydryl groups and the exposure of hydrophobic areas on the surface of the tubulin molecule. To classify the nature of the interaction between halichondrin B and tubulin, in this study we examined the effects of halichondrin B and its closely related analogue, homohalichondrin B, by these assays. We found that: (1) halichondrin B and homohalichondrin B both inhibited formation of an intra-chain cross-link between two sulfhydryl groups in beta-tubulin, as do phomopsin A, dolastatin 10, maytansine, and vinblastine; (2) halichondrin B resembles maytansine in that it had no effect on alkylation of tubulin sulfhydryl groups by iodoacetamide, unlike phomopsin A, dolastatin 10 and vinblastine, all of which inhibit alkylation; (3) halichondrin B differs from other anti-mitotic drugs in that it enhanced exposure of hydrophobic areas on tubulin; (4) homohalichondrin B, like maytansine and in contrast to phomopsin A, dolastatin 10 and vinblastine, had no effect on exposure of hydrophobic areas; and (5) homohalichondrin B, contrary to maytansine, inhibited alkylation of tubulin sulfhydryl groups in the presence of GTP and MgCl2. In their interactions with the tubulin molecule, halichondrin B and homohalichondrin B appear to have unique conformational effects which differ from those of other drugs and also from the effects of each other as well.

Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain.

Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.

Interaction of dolastatin 10 with bovine brain tubulin.

Dolastatin 10 is an unusual peptide of marine origin which binds to tubulin in the vinblastine/maytansine/phomopsin-binding region and potently inhibits mitosis. Using N,N'-ethylenebis(iodoacetamide) (EBI) and iodo[14C]acetamide as probes for the effects of ligands on the thiol groups of tubulin, we found that dolastatin 10 has effects on the sulfhydryls indistinguishable from those of phomopsin A but quite different from those of vinblastine and maytansine. Using the binding of bis-5,5'-[8-(N-phenyl)aminonaphthalene-1-sulfonic acid] (BisANS) as a measure of tubulin decay, we found that dolastatin 10 resembled phomopsin A in that decay was not detectable by this assay in its presence. Interestingly, both otherwise very different peptides are among the most effective inhibitors of tubulin decay yet discovered.

Investigation of the mechanism of the interaction of tubulin with derivatives of 2-styrylquinazolin-4(3H)-one.

A new class of antimitotic agents, derivatives of 2-styrylquinazolin-4(3H)-one (SQZ), was recently described [J. Med. Chem. 33:1721-1728 (1990)]. Because they appeared to interact at a new ligand binding site on tubulin, we attempted to determine their mechanism of action as inhibitors of tubulin polymerization. Although in initial studies inhibition of colchicine binding was negligible, substantial and competitive inhibition of this reaction could be demonstrated with very short incubation times (less than 5 min), provided that a relatively low colchicine to tubulin ratio was used. The initial apparent failure to inhibit colchicine binding resulted from extremely rapid binding to tubulin and dissociation from tubulin by the SQZ derivatives, in comparison with the slow, temperature-dependent, poorly reversible binding of colchicine. The most inhibitory of the SQZ derivatives in the colchicine binding assay was 6-methyl-2-styrylquinazolin-4(3H)-one (NSC 379310), and its interaction with tubulin, particularly as an inhibitor of colchicine binding, was compared with that of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (MTPT), because the binding parameters of MTPT with tubulin have been well described. The data indicate that NSC 379310 binds to tubulin and dissociates from the protein about 3 times as rapidly as MTPT. The other SQZ derivatives with equal or greater potency as inhibitors of tubulin polymerization but apparently less potency as inhibitors of colchicine binding presumably bind to and/or dissociate from tubulin even more rapidly than does NSC 379310.

Tubulin sulfhydryl groups as probes and targets for antimitotic and antimicrotubule agents.

The sulfhydryl groups of tubulin are highly reactive entities. The reactivity of the sulfhydryl groups is sensitive to the presence of tubulin ligands, making these groups excellent probes for the interaction of tubulin with ligands. When tubulin is reacted with N,N'-ethylenebis-(iodoacetamide), two intrachain cross-links form in the beta subunit. Formation of one of these cross-links is completely blocked by colchicine, podophyllotoxin, and nocodazole; formation of the other is blocked completely by maytansine, phomopsin A and GTP and partly by Vinca alkaloids. Different ligands also differ in their effect on the rate of alkylation of tubulin with iodo[14C]acetamide, with vinblastine and phomopsin A being strong inhibitors and maytansine having very little effect. Oxidation of certain key sulfhydryl groups can inhibit microtubule assembly. One of these sulfhydryl groups appears to be cys239, but there are others not yet identified. Sulfhydryl-oxidizing agents also interfere with microtubule-mediated processes in vivo, raising the question of the existence of a physiological regulator of microtubule assembly. Potential physiological regulators have been examined to see if they can control microtubule assembly in vitro at their physiological concentrations. Of the ones that have been examined, thioredoxin and thioredoxin reductase are much better candidates for being physiological regulators than are either cystamine or glutathione.

Interaction of rhizoxin with bovine brain tubulin.

Rhizoxin is an antitumor drug prepared from the fungus Rhizopus chinensis. It is an inhibitor of microtubule assembly and a potent competitive inhibitor of the binding of tubulin of ansamitocin P-3, a maytansine analogue. Rhizoxin also weakly inhibits vinblastine binding to tubulin. We have previously found that maytansine and vinblastine differ strikingly from each other in many ways, including their effects on tubulin sulfhydryl groups and on tubulin decay. Since the structure of rhizoxin is very different from that of vinblastine and only slightly resembles that of maytansine, we decided to compare its interaction with tubulin with those of the other two drugs, using systems which discriminate between the effects of the latter two drugs. We found that rhizoxin acts like maytansine in that it completely prevents formation of an intrachain cross-link in beta-tubulin by N,N'-ethylenebis(iodoacetamide), whereas vinblastine only partially inhibits this. Half-maximal inhibition of formation of this cross-link was observed at 2.5 microM rhizoxin. We found previously that the rate of binding of tubulin to the fluorescent probe bis(8-anilinonaphthalene 1-sulfonate) is a good indicator of tubulin decay and that vinblastine strongly inhibits this, whereas maytansine has no effect. We here report that rhizoxin acts like maytansine in that it has no effect on decay. Thus, despite the fact that its resemblance to maytansine is small, rhizoxin appears to interact with tubulin in very much the same way as does maytansine.

Effect of phomopsin A on the alkylation of tubulin.

Phomopsin A, a macrocyclic heptapeptide isolated from the fungus Phomopsis leptostromiformis, is a potent inhibitor of microtubule assembly and of vinblastine binding to tubulin. Like vinblastine, phomopsin A stabilizes colchicine binding to tubulin. Because phomopsin A is structurally very different from either vinblastine or maytansine, it was of interest to compare its effects on tubulin sulfhydryls to those of the other two drugs. Our results indicate that the effects of phomopsin A combine those of maytansine and vinblastine. Like maytansine, phomopsin A completely inhibited formation of a covalent cross-link between cysteines 12 and 201 or 211, induced by N,N'-ethylenebis(iodoacetamide); like vinblastine, phomopsin A strongly inhibited alkylation of tubulin by iodo[14C]acetamide. Our results are consistent with the hypothesis that phomopsin A binds to regions on tubulin overlapping those to which vinblastine and maytansine bind. We have shown previously that phomopsin A is a potent stabilizer of the tubulin molecule. We now find that when both phomopsin A and colchicine are bound to tubulin, the rate of decay of colchicine binding becomes insignificant.

Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin.

Tubulin, the major constituent protein of microtubules, is a heterodimer of alpha and beta subunits. Both alpha and beta exist in multiple isotypic forms. It is not clear if different isotypes perform different functions. In order to approach this question, we have made a monoclonal antibody specific for the beta III isotype of tubulin. This particular isotype is neuron-specific and appears to be phosphorylated near the C terminus. We have used immunoaffinity depletion chromatography to prepare tubulin lacking the beta III subunit. We find that removal of the beta III isotype results in a tubulin mixture able to assemble much more rapidly than is unfractionated tubulin when reconstituted with either of the two microtubule-associated proteins (MAPs), tau or MAP 2. Our results suggest that the different isotypes of tubulin differ from each other in their ability to polymerize into microtubules. We have also found that the anti-beta III antibody can stimulate microtubule assembly when reconstituted with tubulin and either tau or MAP 2. When reconstituted with tubulin lacking the beta III isotype, the antibody causes the tubulin to polymerize into a polymer that is a microtubule in the presence of MAP 2 and a ribbon in the presence of tau.

The interaction of phomopsin A with bovine brain tubulin.

Phomopsin A is an anti-mitotic compound from the fungus Phomopsis leptostroniformis which is a potent inhibitor of microtubule assembly in vitro; like maytansine, it is known to compete with vinblastine for binding to tubulin (E. Lacey, J. A. Edgar, and C. C. J. Culvenor (1987) Biochem. Pharmacol. 36, 2133-2138). A major difference between the effects of maytansine and vinblastine is that vinblastine is a potent inhibitor of tubulin decay, whereas maytansine has little or no effect on decay. Since phomopsin A is structurally distinct from either maytansine or vinblastine, tubulin decay may be measured by either the time-dependent loss of the ability to bind to [3H]colchicine or the time-dependent increase in the binding of bis(8-anilinonaphthalene 1-sulfonate) (BisANS) to tubulin. By either method, phomopsin A was found to be a much stronger inhibitor of tubulin decay than is vinblastine or any other drug yet tested, and in fact, when decay is measured by the increase of BisANS binding, phomopsin A appears to stop the process entirely. This may prove to be useful in the determination of the higher-order structure of the tubulin molecule.

Determination of methotrexate and its major metabolite, 7-hydroxymethotrexate, using capillary zone electrophoresis and laser-induced fluorescence detection.

High-dosage methotrexate therapy requires careful monitoring of the drug in serum to ensure minimal toxic effects. A simple, rapid and sensitive method for the separation and quantitation of methotrexate and its major metabolite, 7-hydroxymethotrexate, using high-voltage capillary zone electrophoresis combined with laser-induced fluorescence detection is described. The detection limit for methotrexate is as low as 5.10(-10) M (signal-to-noise ratio = 3), while that for 7-hydroxymethotrexate is 2.10(-9) M. The linearity of the system extends over nearly four orders of magnitude for both methotrexate and 7-hydroxymethotrexate. The extraction efficiency for the drug and its metabolite from serum is 80-85% using a Sep-Pak C18 cartridge. Quantitation of methotrexate in serum was possible in the 10(-10) M range, nearly two orders of magnitude lower than that currently obtainable by existing methods. Good correlation (r = 0.99) for serum methotrexate concentrations was obtained with an enzyme-multiplied immunoassay technique. Comparison with an enzyme inhibition assay also provided similar results.