Dislocations dynamics during the nonlinear creep of a homeotropic sample of smectic-A liquid crystal.

New creep experiments under sinusoidal compression/dilation deformation of a homeotropic sample of smectic-A liquid crystal (8CB) show that its response is nonlinear at very small amplitude of deformation. This behavior is explained by taking into account the crossing between the edge dislocations that climb parallel to the layers and the screw dislocations joining the two surfaces limiting the sample. The activation energy of the crossing process and the density of the screw dislocations as a function of the sample thickness are estimated experimentally.

Paclitaxel selects for mutant or pseudo-null p53 in drug resistance associated with tubulin mutations in human cancer.

The efficacy of anticancer therapy is limited by the development of drug resistance. While the role of p53 in the intrinsic sensitivity of human cancer cells to paclitaxel (PTX) remains controversial, its role in acquired paclitaxel resistance has never been addressed. In this study we examined the p53 status of three paclitaxel selected human ovarian carcinoma sublines, resistant to paclitaxel due to acquired beta-tubulin mutations which impair paclitaxel's interaction with tubulin. In contrast to parental cells which have wt p53, in all PTX-resistant sublines p53 was functionally inactive. Two of the resistant sublines expressed high levels of transcriptionally inactive p53 protein, each with a distinct point mutation in codons 236 and 239 of the DNA binding domain. The third subline presented a novel p53 pseudo-null phenotype as a result of markedly decreased wt p53 mRNA expression. Introduction of ectopic wt p53 had no effect on PTX sensitivity in both parental and resistant cells, while it induced p21WAF1/CIP1, demonstrating an intact p53 pathway. While PTX resistance is primarily conferred by the tubulin mutations, the loss of functional p53 observed in all clones, suggests that this loss may facilitate the development of resistance potentially by providing a clonal advantage which promotes the isolation of paclitaxel resistant cells.

A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by tubulin mutations in human cancer cells.

The epothilones are naturally occurring antimitotic drugs that share with the taxanes a similar mechanism of action without apparent structural similarity. Although photoaffinity labeling and electron crystallographic studies have identified the taxane-binding site on beta-tubulin, similar data are not available for epothilones. To identify tubulin residues important for epothilone binding, we have isolated two epothilone-resistant human ovarian carcinoma sublines derived in a single-step selection with epothilone A or B. These epothilone-resistant sublines exhibit impaired epothilone- and taxane-driven tubulin polymerization caused by acquired beta-tubulin mutations (beta274(Thr-->Ile) and beta282(Arg-->Gln)) located in the atomic model of alphabeta-tubulin near the taxane-binding site. Using molecular modeling, we investigated the conformational behavior of epothilone, which led to the identification of a common pharmacophore shared by taxanes and epothilones. Although two binding modes for the epothilones were predicted, one mode was identified as the preferred epothilone conformation as indicated by the activity of a potent pyridine-epothilone analogue. In addition, the structure-activity relationships of multiple taxanes and epothilones in the tubulin mutant cells can be fully explained by the model presented here, verifying its predictive value. Finally, these pharmacophore and activity data from mutant cells were used to model the tubulin binding of sarcodictyins, a distinct class of microtubule stabilizers, which in contrast to taxanes and the epothilones interact preferentially with the mutant tubulins. The unification of taxane, epothilone, and sarcodictyin chemistries in a single pharmacophore provides a framework to study drug-tubulin interactions that should assist in the rational design of agents targeting tubulin.

Mouse Sprr2 genes: a clustered family of genes showing differential expression in epithelial tissues.

Small proline-rich (SPR) proteins are structural components of the cornified cell envelope of stratified squamous epithelia. They are subdivided into three families, i.e., SPR1, SPR2, and SPR3, of which the SPR2 family is the most complex. To understand the significance of this complexity, we have isolated 11 mouse Sprr2 genes, constructed a provisional physical map of the Sprr2 locus on mouse Chromosome 3, and examined the expression patterns of the Sprr2 genes in mouse epithelial tissues. The 11 Sprr2 sequences are highly conserved with a central domain containing a variable number of repeats. In situ hybridization showed the Sprr2 expression to be confined to epithelia. RT-PCR using primers specific for each of the 11 Sprr2 members demonstrated varying degrees of expression among the individual Sprr2 members in different tissues. The correlation between the physical location of the genes in the Sprr2 locus and their expression patterns suggests multiple levels of controlled expression.

Cloning of the gene encoding the murine clathrin-associated adaptor medium chain mu 2: gene organization, alternative splicing and chromosomal assignment.

The mu 2 chain of the clathrin-associated adaptor complex AP-2 is a member of the adaptor medium chain family, a group of proteins involved in the sorting of integral membrane proteins in endocytic/exocytic pathways. Here, we report the cloning of the (MMU)CLAPM1 gene encoding the murine mu 2 chain, the first member of the family for which this information has become available. The mu 2 gene is approximately 8.5 kb long and is organized into 12 exons and 11 introns. Two transcripts are generated by alternative splicing of exon 5, a mini-exon of only six nucleotides. Proteins encoded by both transcripts are capable of interacting with tyrosine-based sorting signals, suggesting that they are functionally equivalent. The mu 2 gene is localized to the proximal region of mouse chromosome 16, which is syntenic to the proximal region of human chromosome 3. The isolation and characterization of the mu 2 gene should be instrumental for future studies of the genetics and physiological role of the adaptor medium chains in mammals.

Altered expression of a novel adaptin leads to defective pigment granule biogenesis in the Drosophila eye color mutant garnet.

Drosophila eye pigmentation defects have thus far been attributed to mutations in genes encoding enzymes required for biosynthesis of pigments and to ABC-type membrane transporters for pigments or their precursors. We report here that a defect in a gene encoding a putative coat adaptor protein leads to the eye color defect of garnet mutants. We first identified a human cDNA encoding delta-adaptin, a structural homolog of the alpha- and gamma-adaptin subunits of the clathrin coat adaptors AP-1 and AP-2, respectively. Biochemical analyses demonstrated that delta-adaptin is a component of the adaptor-like complex AP-3 in human cells. We then isolated a full-length cDNA encoding the Drosophila ortholog of delta-adaptin and found that transcripts specified by this cDNA are altered in garnet mutant flies. Examination by light and electron microscopy indicated that these mutant flies have reduced numbers of eye pigment granules, which correlates with decreased levels of both pteridine (red) and ommachrome (brown) pigments. Thus, the eye pigmentation defect in the Drosophila garnet mutant may be attributed to compromised function of a coat protein involved in intracellular transport processes required for biogenesis or function of pigment granules.

Structural determinants of interaction of tyrosine-based sorting signals with the adaptor medium chains.

Many integral membrane proteins contain tyrosine-based signals within their cytoplasmic domains that mediate internalization from the cell surface and targeting to lysosomal compartments. Internalization depends on an interaction of the tyrosine-based signals with the clathrin-associated adaptor complex AP-2 at the plasma membrane, whereas lysosomal targeting involves interaction of the signals with an analogous complex, AP-1, at the trans-Golgi network. Recent studies have identified the medium chains mu2 of AP-2 and mu1 of AP-1 as the recognition molecules for tyrosine-based signals. We have now investigated the structural determinants for interaction of the signals with mu2 and mu1. The position of the signals was found to be an important determinant of interactions with mu2 and mu1; signals were most effective when present at the carboxyl terminus of a polypeptide sequence. Another important determinant of interactions was the identity of residues surrounding the critical tyrosine residue. Mutation of some residues affected interactions with mu2 and mu1 similarly, whereas other mutations had differential effects. These observations suggest that both the position and the exact sequence of tyrosine-based sorting signals are major determinants of selectivity in their interaction with clathrin-associated adaptor complexes.

Phospholipids stabilize the interaction between the alpha and beta subunits of the solubilized receptor for immunoglobulin E.

The cell-surface component (alpha) which binds monomeric immunoglobulin E with high affinity is associated with a second polypeptide (beta) in the plasma membrane. The latter component tends to dissociate during purification of the alpha chain from detergent extracts of cells, even at neutral pHs and physiological ionic strengths. We now report that the interaction of alpha and beta can be stabilized by maintaining an appropriate phospholipid to detergent ratio. Under such conditions, other discrete components reproducibly copurify with the alpha and beta chains. These results suggest that the subunits of this membrane protein--or the interaction of it with other constituents in the cell--may be stabilized in ways not observed with ordinary soluble proteins.

Unexpected findings from target analysis of immunoglobulin E and its receptor.

The membrane receptor for immunoglobulin E (IgE) and its ligand, IgE, were irradiated with high-energy electrons. Loss of binding activity was measured for each, and the size of the functional targets was assessed. In both cases, the target size was substantially smaller than the covalent structure of the molecule. The direction of this discrepancy is unprecedented on the basis of experience with loss of enzymatic activity by irradiation; indeed, two enzymes which were present in the receptor preparations gave expected values when measured simultaneously. We suggest that in instances where a function such as ligand binding resides in a conformationally stable domain, radiation inactivation may be capable of revealing this.

Composition and subunit structure of the cell receptor for immunoglobulin E.

The principal surface glycoprotein which specifically binds immunoglobulin E was isolated from rat basophilic leukemia cells in sufficient amounts for compositional and end group analyses. The protein has about 30% carbohydrate and a relatively low content of hydrophobic amino acid residues. No NH2-terminal residue was found by standard methods. The data suggest a Mr approximately equal to 50,000. The latter value is calculated on the basis of 1 molecule of receptor binding 1 molecule of immunoglobulin E. New data confirmed this valence. We propose a provisional model in which the principal component which binds immunoglobulin E is a monomer which, in cells and in nondenaturing solvents, is associated in a 1:1 ratio with the polypeptide of Mr approximately equal to 30,000 recently defined by studies employing cross-linking reagents.

Triggering of cultured neoplastic mast cells by antibodies to the receptor for IgE.

Cell surface receptors for IgE were isolated from detergent lysates of iodinated, IgE-saturated, rat basophilic leukemia cells by precipitation with anti-IgE antibodies followed by chromatography at acid pH. The isolated material showed a single 125I-band (m.w. approximately 58,000) on gel electrophoresis in sodium dodecyl sulfate and was used to immunize a rabbit. The resulting anti-serum was reacted with lysates of surface iodinated mouse or rat tumor mast cells. Analysis of the precipitates on (10%) gel electrophoresis revealed one major peak comprising greater than 80% of the detectable counts and having an estimated m.w. of approximately 58,000. The antiserum reacted with detergent-solubilized and cell-bound receptors in the presence or absence of excess IgE; it also inhibited the binding of 125I-IgE. Cultured mouse mastocytoma cells never exposed to IgE released 3H-serotonin when incubated with F(ab')2, but not Fab' fragments of the antiserum, which had been rigorously freed of IgE and anti-IgE. The release was inhibited in the presence of excess IgE, was Ca++ dependent, and equaled 80% of the maximum obtained with IgE and anti-IgE. We conclude that aggregation of the receptors for IgE provides the critical signals for cell activation.

[Bipedal lymphography in mediastino-pulmonary sarcoidosis. Report concerning 9 cases]. / La linfografia bipedidia nella sarcoidosi mediastino-polmonare. Relazione su 9 casi.

The relevant literature is briefly reviewed and lymphographic data obtained from 9 patients admitted to the S. L. Gonzaga Hospital are examined. In all cases, the diagnosis of sarcoidosis had been given prior to lymphography and confirmed histologically. This small series helps to show the extent of lymph node lesions in sarcoidosis and the more commonly observed adenographic features.