Does V600E BRAF mutation predict vinorelbine efficacy? A proof-of-concept from a lung micropapillary adenocarcinoma metastatic to the breast.

BRAF mutations occur in about 3% of all lung adenocarcinomas and V600E missense mutation characterizes about half of BRAF-mutated lung adenocarcinomas and is significantly associated with micropapillary pattern and shorter disease-free and overall survival rates. In this report, we report a challenging case of a patient with a metastatic micropapillary adenocarcinoma of the lung harbouring V600E BRAF mutation who experienced a surprising protracted clinical response to metronomic vinorelbine. The possible association between the V600E BRAF mutation pathway and the effective use of vinca alkaloid is discussed.

A new mutant of Arabidopsis disturbed in its roots, right-handed slanting, and gravitropism defines a gene that encodes a heat-shock factor.

A new mutant of Arabidopsis named rha1 is characterized and the gene involved cloned. In roots, the mutant shows minimal right-handed slanting, reduced gravitropic response, notable resistance to 2,4-D, but scarce resistance to IAA and NAA. The roots also show a clear resistance to the auxin transport inhibitors TIBA and NPA, and to ethylene. Other characteristics are a reduced number of lateral roots and reduced size of shoot and root in the seedlings. The gene, cloned through TAIL-PCR, was found to be a heat-shock factor that maps on chromosome 5, close to and above the RFLP marker m61. The rha1 structure, mRNA, and translation product are reported. Since, so far, no other gravitropic mutant has been described as mutated in a heat-shock factor, rha1 belongs to a new group of mutants disturbed in slanting, gravitropism, and auxin physiology. As shown through the RT-PCR analyses of its expression, the gene retains the function connected with heat shock. If the characteristics connected with auxin physiology are considered, however, it is also likely that the gene, as a transcription factor, could be involved in root circumnutation, gravitropic response, and hormonal control of differentiation. Since GUS staining under the gene promoter was localized mainly in the mature tissues, rha1 does not seem to be involved in the first steps of gravitropism, but is rather related to the general response to auxin. The alterations in slanting (mainly due to reduced chiral circumnutation) and gravitropism lead to the supposition that the two processes may have, at least in part, common origins.

Chiral and non-chiral nutations in Arabidopsis roots grown on the random positioning machine.

Arabidopsis thaliana roots grown on a vertically set plate do not elongate straight down the gravitational vector, but by making waves and coils, and by conspicuously slanting towards the right-hand. This behaviour, in a previous paper, was ascribed to the simultaneous effect of three processes: circumnutation, positive gravitropism and negative thigmotropism. However, when the plants are grown on the Random Positioning Machine (RPM), in conditions that are believed to simulate space microgravitational conditions closely, the roots do not show the usual pattern. In the wild type, the roots make large loops to the right-hand side, whereas in the gravitropic and auxinic mutants aux1, eir1, rha1, they just move randomly around the initial direction. Therefore, if the movements made on the RPM are those produced by the exclusion of gravitropism and negative thigmotropism, as is apparent, the conclusion is that Arabidopsis roots are animated by a form of chiral circumnutation, that is lacking in the auxinic and gravitropic mutants aux1, eir1 and rha1. In addition, the 1 g condition appears to reduce the scatter among the circumnutating tracks produced by the roots of the wild types, but not among those of the mutants. Because there is a scarcity of literature regarding circumnutation in roots, it is not known how widely root chiral circumnutation is spread, but it is known that, in some previously studied species, just random nutations are observed. Two kinds of nutating movements seem to exist in plant roots and, whereas the random process does not seem to be connected with auxin physiology and transport, the chiral process appears to be connected in the same way as gravitropism is.

Spiralizations and tropisms in Arabidopsis roots.

When Arabidopsis seedlings are grown on a hard-agar plate, their primary roots show characteristic spiralling movements, apparent as waves, coils and torsions, together with a slanting toward the right-hand side. All these movements are believed to be the result of three different processes acting on the roots: circumnutation, positive gravitropism and negative thigmotropism. The basic movement of the roots is described as that of a growing right-handed helix, which, because of the root tip hitting the agar plate, is continuously switched from the right-hand to the left-hand of the growth direction, and vice versa. This movement also produces a slanting root-growth direction toward the right-hand because of the incomplete waves made by the right-handed root to the left-hand. By contrast, the torsions seen in the coils and waves are interpreted as artefacts that form as an adaptation of the three-dimensional root helix to the flat two-dimensional agar surface.

A new Arabidopsis thaliana root gravitropism and chirality mutant.

A new Arabidopsis thaliana (L.) Heynh. root mutant, named clg1, was isolated from the Feldmann-Du Pont T-DNA insertional mutagenesis collection. It is characterized by primary roots that make early right-handed coils, show increased right-handed slanting, and reduced positive gravitropism. The mutant roots however did not exhibit increased resistance to IAA, but only a modest increment of resistance to the auxins 2,4 D, and NAA, and to the auxin transport inhibitors TIBA and NPA. By contrast, the mutant roots showed a notable resistance to plant hormone ethylene (given as ACC). clg1 appears to be new, since it complements the most known auxin and gravitropism mutants, maps to chromosome 5, and shows a phenotype largely different from that of the known ethylene mutants. The increased right-handed slanting (chirality) can possibly be a consequence of the reduced gravitropic response, since gravitropism and slanting are competitive growth-direction leading forces. The increased resistance to ethylene, seems to indicate that this phythormone plays a role in the gravitropic response of roots (as already proposed for shoots), and possibly in the regulation of the connected signal transduction pathway. The gene involved in the clg1 mutation, which is recessive, was mapped, as above reported, to chromosome 5, close to the visible marker tt3.

The right-handed slanting of Arabidopsis thaliana roots is due to the combined effects of positive gravitropism, circumnutation and thigmotropism.

When primary Arabidopsis roots grow down a tilted agar plate, they do not elongate following the gravitational vector along a straight line, but instead they slant noticeably to the right-hand. This process is seen mostly in the ecotypes Wassilewskjia and Landsberg, whereas it is attenuated in the ecotype Columbia, and in some mutants is even inverted. The origin of the slanting of Arabidopsis roots, that evidently constitutes a form of chirality, has so far not been sufficiently clarified. In the present paper we describe it as the general result of the cumulative effects of positive gravitropism, circumnutation and a thigmotropic obstacle-avoiding movement, and in particular, as the consequence of the alternating movement of circumnutation of the root to the right and to the left of the gravitational vector. This movement, which does not appear symmetrical in its nature, since the waves made to the right-hand are complete whereas those made to the left-hand are reduced or aborted, appears to be the reason for the observed slanting. In addition, evidence is furnished supporting the hypothesis that the strong left-handed cell-files torsion, seen in right-handed coiling roots, is not the consequence of a primary process, but of an artifact, and is due to the adjustment of the three dimensional root circumnutating helix to the flat two dimensional agar surface.

Understanding the gravitropic signal transduction pathway through the analysis of new Arabidopsis mutants.

The gravitropic response of plants can be divided into three steps: detection, transduction, and asymmetric growth. Of these the second is that on which we have the smallest knowledge. Even though we have in fact some support for a redistribution of auxin as the agent of the graviresponse (Cholodny-Went hypothesis), we still lack of a precise knowledge about the many steps which precede and follow auxin redistribution. We thereore today dispose of the powerful new techniques of molecular genetics, that can help us in dissecting the pathway. By these techniques, as is known, already a number of gravitropic mutants have been isolated in the model plant Arabidopsis, and, in addition, some of the genes involved have been cloned. These findings already permit some speculation about the gravitropic pathway, even though we still are far from an understanding of the process. With the aim thus of obtaining the information we need to elucidate the gravitropic pathway, in the last two years, we performed a screening of some significant collections of Arabidopsis mutants, isolating individuals disturbed in root gravitropism, and starting to clone the involved genes.

Copresence of Deleted Protein Species Generates Structural Heterogeneity of Ovine alpha(s1)-Casein.

Multiple forms of mature alpha(s1)-casein have been characterized in ovine variants A and D using a combination of mass spectrometry and automated Edman degradation. Mature ovine alpha(s1)-casein was found to be a heterogeneous mixture of at least seven molecular species. The main component, representing about 50% total alpha(s1)-casein, corresponded to the full-length (199 residues long) protein. The other components were alpha(s1)-casein of different lengths: 198 (less Gln78), 191 (less peptide 110-117), 191 residues (less peptide 140-148), 190 (less peptide 110-117 and Gln78), 190 (less peptide 140-148 and Gln78), and 183 (less peptides 110-117 and 140-148) residues long alpha(s1)-casein. Each of the alpha(s1)-casein multiple forms occurred at three different phosphorylation levels, due to the partial phosphorylation of both Ser115 (at about 50%) and Ser41 (at about 20%). In the case of deleted peptide 110-117, the protein heterogeneity linked to the partially phosphorylated Ser115 was abolished, and only two levels of phosphorylation were observed. These multiple forms differing in molecular weight and degree of phosphorylation may have been developed from an exon skipping during mRNA splicing in ovine alpha(s1)-casein, similar to that recently described in the case of its caprine counterpart.

A novel root gravitropism mutant of Arabidopsis thaliana exhibiting altered auxin physiology.

A root gravitropism mutant was isolated from the DuPont Arabidopsis thaliana T-DNA insertional mutagenesis collection. This mutant has reduced root gravitropism, hence the name rgr1. Roots of rgr1 are shorter than those of wild-type, and they have reduced lateral root formation. In addition, roots of rgr1 coil clockwise on inclined agar plates, unlike wild-type roots which grow in a wavy pattern. The rgr1 mutant has increased resistance, as measured by root elongation, to exogenously applied auxins (6-fold to indole-3-acetic acid, 3-fold to 2,4-dichlorophenoxyacetic acid, and 2-fold to napthyleneacetic acid). It is also resistant to polar auxin transport inhibitors (2-fold to triiodobenzoic acid and 3- to 5-fold to napthylphthalamic acid). The rgr1 mutant does not appear to be resistant to other plant hormone classes. When grown in the presence of 10(-7) M 2,4-dichlorophenoxyacetic acid, rgr1 roots have fewer root hairs than wild type. All these rgr1 phenotypes are Mendelian recessives. Complementation tests indicate that rgr1 is not allelic to previously characterized agravitropic or auxin-resistant mutants. The rgr1 locus was mapped using visible markers to 1.4 +/- 0.6 map units from the CH1 locus at 1-65.4. The rgr1 mutation and the T-DNA cosegregate, suggesting that rgr1 was caused by insertional gene inactivation.

Preparation, characterization, and evaluation of a monoclonal antibody against the rabbit platelet glycoprotein IIb/IIIa in an experimental angioplasty model.

The deposition of platelets at the site of balloon angioplasty is thought to play a major role in the pathogenesis of restenosis. The antibody AZ-1, which binds to the rabbit platelet glycoprotein IIb/IIIa receptor and inhibits platelet function both in vitro and in vivo, was produced and tested in an experimental model of angioplasty. Atherosclerosis was induced by desiccation injury of the femoral artery, followed by a 28-day diet with 2% cholesterol and 6% peanut oil. Rabbits were randomized to receive an infusion of saline, a single infusion of 0.5 mg/kg of AZ-1, or an infusion of 0.6 mg/kg AZ-1 before angioplasty. The latter group received a second infusion of 0.6 mg/kg 72 hours later. Functional platelet inhibition was demonstrated by prolongation of the bleeding time in all treated animals. Angiography was performed at baseline, immediately after a standardized angioplasty, and again 28 days after angioplasty on a total of 42 vessels. There were no significant differences between the antibody-treated group and the control group in the mean angiographic minimum luminal diameter at any of the time points. There was also no difference in the initial improvement after angioplasty (acute gain), in the decrease in luminal diameter from immediately after angioplasty to 28 days after angioplasty (late loss), or in the overall improvement from before angioplasty to 28 days after angioplasty. Quantitative histological analysis confirmed the lack of a beneficial effect of AZ-1. There were no significant differences in the area of the intima, the media, or the combined intima and media between the antibody-treated groups and the control group. Thus, potent platelet inhibition for up to 6 days after balloon angioplasty using a monoclonal antibody that inhibits platelet aggregation did not reduce the response to vascular injury after balloon angioplasty in this rabbit model of experimental atherosclerosis.

A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries.

Tissue factor (TF) is a transmembrane protein that binds factor VII/VIIa, thus activating the extrinsic blood coagulation pathway. Since this pathway appears to be involved in the formation of intravascular thrombi, the anti-rabbit TF monoclonal antibody, AP-1, was produced and tested as an antithrombotic agent in a rabbit model of recurrent intravascular thrombosis. In this model, a plastic constrictor is positioned around the injured rabbit carotid arteries, and flow is monitored with a Doppler flow probe. This produces cyclic flow variation (CFV) in the carotid artery, which is caused by recurrent formation and dislodgment of thrombi at the site of the stenosis. After monitoring CFV pattern for 30 minutes, AP-1 was infused intravenously into nine rabbits at doses of 0.05 to 1.5 mg/kg body weight, and a control monoclonal antibody that does not react with rabbit TF was infused into four additional rabbits. In all rabbits receiving AP-1, CFV was abolished, and a steady normal blood flow was restored, indicating that thrombus formation had been blocked by AP-1. By contrast, in all rabbits that received the control monoclonal antibody, CFV continued unaltered. There was no change in the partial thromboplastin time and ex vivo platelet aggregation to several different agonists after infusion of AP-1, indicating an absence of systemic effects on the coagulation process. We conclude that activation of the extrinsic coagulation pathway has a key role in triggering intravascular thrombosis and that an anti-TF monoclonal antibody is an effective antithrombotic agent that could have therapeutic potential for humans.

Molecular analysis of three maize 22 kDa auxin-binding protein genes--transient promoter expression and regulatory regions.

The site I 22 kDa auxin-binding proteins from maize are encoded by a small gene family comprising at least five members. Here the cloning and molecular analysis of the Zm-ERabp1, Zm-ERabp4, and Zm-ERabp5 genes is presented. All three encode 22-23 kDa proteins displaying a transit peptide, a C-terminal KDEL sequence, as well as glycosylation and auxin-binding sites. The Zm-ERabp4 and Zm-ERabp5 genes are very similar. The Zm-ERabp1 gene encodes a related protein, but its promoter, leader and signal peptide are very different. Northern analysis using gene-specific oligonucleotide probes indicates that Zm-ERabp4 is expressed in leaves and coleoptiles but weakly in roots, whereas Zm-ERabp5 expression is barely detectable in these tissues. RNA-PCR indicated that all three genes are none the less expressed in many tissues. Primer-extension analysis revealed an unusually long (320 bases) Zm-ERabp1 leader containing an 80 codon ORF which, if expressed, would encode a positively charged protein with some similarity to transcription factors. In a transient promoter-reporter gene expression system using maize leaf protoplasts the Zm-ERabp1 promoter is more active than the Zm-ERabp4 and Zm-ERabp5 promoters. Promoter deletion analysis of Zm-ERabp1 has identified a negative regulatory sequence in a region from -364 bp and -130 bp, deletion of which results in about twofold higher expression. This region contains both enhancer- and G-box-related sequences. Deletion of -126 bp to +64 bp, which contains the TATA box and transcription start, results in a large decrease in expression.

Comparison of platelet scintigraphy, impedance plethysmography gray scale and color flow duplex ultrasound and venography for the diagnosis of venous thrombosis.

The several techniques available for the diagnosis of venous thrombosis have not been directly compared in the same patient population. Thus color and gray scale duplex ultrasound (U), impedance plethysmography (IPG), 3-4 hr platelet imaging (PS) were compared to venography (V), in 104 consecutive patients (in hospital and out). PS and V were read by two, and IPG and U by one, blinded reader. Comparisons were made for the calf (CA), popliteal (Pop) and femoral (Fem) vessels. Reproducibility of V and PS was 84 and 87%. (table; see text) We conclude that PS, while having a very high specificity, has an unacceptably low sensitivity. However, while both impedance plethysmography and color flow ultrasound have excellent and similar diagnostic accuracy in the femoral, these techniques have either a low sensitivity or low technical success rate in the calf or popliteal veins.

Effect of asymmetric auxin application on Helianthus hypocotyl curvature.

Indole-3-acetic acid was applied asymmetrically to the hypocotyls of sunflower (Helianthus annuus L.) seedlings. After 5 hours on a clinostat, auxin gradients as small as 1 to 1.3 produced substantial (more than 60 degrees) hypocotyl curvature. This result suggests the asymmetric growth underlying hypocotyl gravitropism can be explained by lateral auxin redistribution.

On the role of calcium in indole-3-acetic acid movement and graviresponse in etiolated pea epicotyls.

To determine whether Ca2+ plays a special role in the early graviresponse of shoots, as has been reported for roots, we treated etiolated pea epicotyls with substances known to antagonize Ca2+ (La3+), to remove Ca2+ from the wall (spermidine, EGTA), to inhibit calmodulin mediated reactions (chlorpromazine), or to inhibit IAA transport (TIBA). We studied the effect of these substances on IAA and Ca2+ uptake into 7 mm long subapical 3rd internode etiolated pea epicotyl sections and pea leaf protoplasts, on pea epicotyl growth, and graviresponse and on lateral IAA redistribution during gravistimulation. Our results support the view that adequate Ca2+ in the apoplast is required for normal IAA uptake, transport and graviresponse. Experiments with protoplasts indicate that Ca2+ may be controlling a labile membrane porter, possibly located on the external surface of cell membrane, while inhibitor experiments suggest that calmodulin is also implicated in both the movement of IAA and graviresponse. Since a major transfer of Ca2+ through free space during graviresponse has not yet been demonstrated, and since inhibition of calcium channels does not affect IAA redistribution (Migliaccio and Galston, 1987, Plant Physiology 85:542), we conclude that no clear evidence links prior Ca2+ movement with IAA redistribution during graviresponse in stems.

On the nature and origin of the calcium asymmetry arising during gravitropic response in etiolated pea epicotyls.

Seven day old etiolated pea epicotyls were loaded symmetrically with 3H-indole 3-acetic acid (IAA) or 45Ca2+, then subjected to 1.5 hours of 1g gravistimulation. Epidermal peels taken from top and bottom surfaces after 90 minutes showed an increase in IAA on the lower side and of Ca2+ on the upper side. Inhibitors of IAA movement (TIBA, 9-hydroxyfluorene carboxylic acid) block the development of both IAA and Ca2+ asymmetries, but substances known to interfere with normal Ca2+ transport (nitrendipine, nisoldipine, Bay K 8644, A 23187) do not significantly alter either IAA or Ca2+ asymmetries. These substances, however, are active in modifying both Ca2+ uptake and efflux through oat and pea leaf protoplast membranes. We conclude that the 45Ca2+ fed to pea epicotyls occurs largely in the cell wall, and that auxin movement is primary and Ca2+ movement secondary in gravitropism. We hypothesize that apoplastic Ca2+ changes during graviresponse because it is displaced by H+ secreted through auxin-induced proton release. This proposed mechanism is supported by localized pH experiments, in which filter paper soaked in various buffers was applied to one side of a carborundum-abraded epicotyls. Buffer at pH 3 increases calcium loss from the side to which it is applied, whereas pH 7 buffer decreases it. Moreover, 10 micromolar IAA and 1 micromolar fusicoccin, which promote H+ efflux, increase Ca2+ release from pea epicotyl segments, whereas cycloheximide, which inhibits H+ efflux, has the reverse effect. We suggest that Ca2+ does not redistribute actively during gravitropism: the asymmetry arises because of its release from the wall adjacent to the region of high IAA concentration, proton secretion, and growth. Thus, the asymmetric distribution of Ca2+ appears to be a consequence of growth stimulation, not a critical step in the early phase of the graviresponse.

Sequence of key events in shoot gravitropism.

It has recently been shown that asymmetric acid efflux is closely correlated with the gravitropic curvature of plant shoots and roots. The research reported here addresses whether auxin (IAA) redistribution in shoots is the cause or result of asymmetric acid efflux.When abraded sunflower (Helianthus annuus cv Mammoth) hypocotyls are submerged in 20 millimolar neutral buffer, gravicurvature is greatly retarded relative to 0.2 millimolar controls. Nevertheless, in both buffer systems there is a similar redistribution of [(3)H]IAA toward the lower surface of gravistimulated sunflower hypocotyls. These results suggest that graviperception initiates IAA redistribution, which in turn results in auxin-induced asymmetric H(+) efflux across the shoot. This interpretation is reinforced by data showing the effects of removal of the epidermal layers (peeling), osmotic shock, and morphactin treatment on gravicurvature and [(3)H]IAA redistribution. Peeling and osmotic shock inhibit gravicurvature but not redistribution. Morphactin inhibits both processes but does not inhibit hypocotyl straight growth.