Inhibition of cholesteatoma migration in vitro with all-trans retinoic acid.

Retinoids have recently become of interest to clinicians because of their ability to inhibit migration and proliferation of premalignant squamous cells while enhancing growth and proliferation of normal cells. An in vitro investigation was undertaken to determine whether retinoic acid exhibits similar inhibitory effects on cholesteatoma cells. Cholesteatoma specimens were obtained intraoperatively from 10 patients undergoing mastoidectomy or revision mastoidectomy for chronic ear disease.

Significant divergence in nucleotide sequences for beta-tubulin from different laboratory strains of Chinese hamster ovary cells.

The nucleotide sequences for isotype 1 beta-tubulin cDNAs cloned from different laboratory strains of Chinese hamster ovary (CHO) cells were compared and found to contain an unexpected number of sequence differences in both translated and untranslated regions of the gene. The results indicate significant changes in the DNA, but not protein, sequence while the cells have been in culture and reveal sequences in the 5' and 3' untranslated regions that have resisted these changes.

Overexpression of an epitope-tagged beta-tubulin in Chinese hamster ovary cells causes an increase in endogenous alpha-tubulin synthesis.

A Chinese hamster beta-tubulin cDNA, engineered to express a 9 amino acid epitope from the influenza hemagglutinin antigen (HA), was transfected into Chinese hamster ovary (CHO) cells. The recombinant protein (HA beta 1-tubulin) appeared to behave normally by the following criteria: immunofluorescence indicated that HA beta 1-tubulin incorporated into all classes of interphase and spindle microtubules as well as microtubule organizing centers. The sensitivity of the cells expressing HA beta 1-tubulin to Colcemid and taxol was unchanged.

[Chronic phase of Chagas disease. Clinical aspects and course of the disease].

Purpose: To evaluate the clinical and evolutive aspects of chronic chagasic patients.

Methods: Three hundred chronic chagasic patients, 180 females, with age ranging from 19 to 81 years (55.6 +/- 13.

Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization.

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate.

Estramustine depolymerizes microtubules by binding to tubulin.

To investigate the mechanism of action of the antineoplastic drug estramustine, we compared its effects on human prostate cancer cells with those of vinblastine. At their respective concentrations that result in 50% inhibition of clonogenic growth, both drugs caused an accumulation of cells blocked at mitosis and similar dose- and time-dependent depolymerization of interphase microtubules. Also, colcemid-resistant and colcemid-hypersensitive Chinese hamster ovary cells with tubulin mutations were collaterally cross-resistant or -sensitive to estramustine.

Use and environmental implications of pulp-mill sludge as an organic fertilizer.

Pulp-mill sludge is an industrial by-product with a high level of organic matter, which is eventually biodegradable. Since there are few alternatives for its practical use, its utilization as an agricultural fertilizer is proposed. An experiment was carried out during 2 years, with yellow lupin plants (Lupinus luteus L.

[Civil aviation and cardiology. Admission rules and follow-up of the technical flying personnel of TAP-Air Portugal].

The authors, based in European and American rules, consensus positions of Clinical Aerospace Congresses and their own experience, marked admission and follow-up rules of conduct for TAP Air Portugal aircrew. They stressed the importance of modern technology in arterial pressure ambulatory diagnosis and pointed the necessity of arterial pressure treatment in the other cardiovascular risk factors context. They relief ischemic myocardial disease because it is incompatible with flying safety, even in those submitted to coronary angioplasty or bypass graft surgery.

Resistance to antimitotic agents as genetic probes of microtubule structure and function.

Much of our knowledge about microtubules has come from detailed morphological, biochemical, and cell biological studies. As more is learned about these organelles, questions regarding the in vivo regulation of their expression and function become increasingly important. Genetics provides an approach to address these more subtle questions in the living cell.

Resistance to antimitotic drugs in Chinese hamster ovary cells correlates with changes in the level of polymerized tubulin.

A sensitive and reproducible method to measure relative levels of polymerized and soluble tubulin in cells has been developed. This method involves metabolically labeling cells with radioactive amino acids followed by lysis in a microtubule-stabilizing buffer, centrifugation to separate soluble from polymerized tubulin, resolution of the proteins in each fraction by two-dimensional gel electrophoresis, and quantitation of the tubulin by liquid scintillation counting of spots excised from the gel. Several buffers were evaluated for their reproducibility and efficacy in preserving the state of in vivo microtubule assembly at the time of cell lysis, and the ability of the technique to measure drug-induced changes in tubulin polymerization was determined.

Alterations in microtubule assembly caused by the microtubule-active drug LY195448.

LY195448 is an experimental drug that blocks cells at metaphase (Boder et al.: Microtubules and Microtubule Inhibitors 1985: 353-361, 1985). A 4 hour exposure of NRK cells to a drug concentration of 46 microM (15 micrograms/ml) increased the number of mitotic cells in the population from 4.

Identification of methionine-containing tryptic peptides of unstable beta-tubulin separated by reverse-phase high-performance liquid chromatography.

Methods for examining altered regions in unstable mutant proteins are described. The strategy is illustrated using assembly defective Chinese hamster beta-tubulin subunits that are rapidly degraded in the cell. These unstable proteins are metabolically labeled to high specific activity and isolated as spots on two-dimensional gels.

Mechanisms by which mammalian cells acquire resistance to drugs that affect microtubule assembly.

The development of resistance in mammalian cells to toxic drugs is a significant clinical problem, especially in cancer chemotherapy where drug-resistant tumor cells often prove to be refractory to treatment. In this article, we review some of the basic mechanisms of drug resistance from the perspective of a single cell bathed in medium containing the drug. These mechanisms may be categorized according to changes in the cell that affect the ability of the drug to accumulate intracellularly, changes in enzymes that are required for drug toxicity, alterations in trapping of the drug or detoxification of the drug, alterations in binding to an intracellular target, or alterations in cellular processes that compensate for the action of the drug.

Expression and function of beta-tubulin isotypes in Chinese hamster ovary cells.

The availability of isotype-specific antisera for beta-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of beta-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor beta-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype-specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V.

Elimination of permeability mutants from selections for drug resistance in mammalian cells.

Chinese hamster ovary (CHO) cells exhibit increased sensitivity to a wide variety of microtubule inhibitory drugs when verapamil is present in the growth medium. The extent of this increased sensitivity is drug specific: some drugs such as taxol and vinblastine respond greatly to the presence of verapamil, whereas other drugs such as griseofulvin respond very poorly. For the majority of drugs examined, however, a 2- to 10-fold increase in drug sensitivity is observed in the presence of verapamil at 5 micrograms/ml.

Contact sensitivity to oak moss.

Oak moss allergy was the principle allergen in contact sensitivity to perfumes (45%); 31 patients reacting to oak moss were studied. The sensitivity was attributed to contact with perfumes in 20, lichens in 7 and unknown in 4. Atranorin was the most frequent allergen, followed by usnic, evernic and fumarprotocetraric acids.

Mutations affecting assembly of beta-tubulin localize to a region near the carboxyl terminus.

The generation of Chinese hamster ovary cell lines that express assembly defective forms of beta-tubulin were isolated using selections based on reversion of conditional lethal or drug resistance phenotypes. Two such cell lines, D2 and 6H3, were chosen for further characterization because they contain beta-tubulin polypeptides that exhibit decreases in apparent molecular weight on two-dimensional gel electrophoresis. Analysis of the nucleic acid from these cell lines using both Southern and Northern procedures suggests a deletion in one of the beta-tubulin genes in each cell line.

Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells.

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E.

Biochemical characterization of isolated CHO cell mitotic spindles: identification of calmodulin-binding proteins.

Mitotic spindles were isolated from Chinese hamster ovary (CHO) cells and examined morphologically and biochemically. The isolated spindles were observed to be intact structures containing associated chromosomes and were surrounded by a cage of vimentin-containing filaments. Two-dimensional gel electrophoresis of isolated spindles versus whole cell homogenates indicated that isolated spindles were free from significant cytoplasmic contamination and contained tubulin, actin, vimentin and an 80 X 10(3) Mr quadrapeptide as their major protein constituents.

Identification of a 52-kD calmodulin-binding protein associated with the mitotic spindle apparatus in mammalian cells.

A pool of 10 calmodulin-binding proteins (CBPs) was isolated from Chinese hamster ovary (CHO) cells via calmodulin (CaM)-Sepharose affinity chromatography. One of these ten isolated CBPs with a molecular mass of 52 kD was also found to be present in isolated CHO cell mitotic spindles. Affinity-purified antibodies generated against this pool of isolated CBPs recognize a single 52-kD protein in isolated CHO cell mitotic spindles by immunoblot analysis.

Taxol-dependent mutants of Chinese hamster ovary cells with alterations in alpha- and beta-tubulin.

Chinese hamster ovary cell mutants resistant to the microtubule stabilizing drug taxol were isolated in a single step. Of these 139 drug-resistant mutants, 59 exhibit an absolute requirement for taxol for normal growth and division, 13 have a partial requirement, and 69 grow normally without the drug. Two-dimensional gel analysis of whole cell proteins reveal "extra" spots representing altered tubulins in 13 of the mutants.