Present and future breast cancer management--bench to bedside and back: a positioning paper of academia, regulatory authorities and pharmaceutical industry.

Insights into tumour biology of breast cancer have led the path towards the introduction of targeted treatment approaches; still, breast cancer-related mortality remains relatively high. Efforts in the field of basic research revealed new druggable targets which now await validation within the context of clinical trials. Therefore, questions concerning the optimal design of future studies are becoming even more pertinent. Aspects such as the ideal end point, availability of predictive markers to identify the optimal cohort for drug testing, or potential mechanisms of resistance need to be resolved. An expert panel representing the academic community, the pharmaceutical industry, as well as European Regulatory Authorities met in Vienna, Austria, in November 2012, in order to discuss breast cancer biology, identification of novel biological targets and optimal drug development with the aim of treatment individualization. This article summarizes statements and perspectives provided by the meeting participants.

Calcium-signaling networks in olfactory receptor neurons.

The olfactory neuroepithelium represents a unique interface between the brain and the external environment. Olfactory function comprises a distinct set of molecular tasks: sensory signal transduction, cytoprotection and adult neurogenesis. A multitude of biochemical studies has revealed the central role of Ca(2+) signaling in the function of olfactory receptor neurons (ORNs). We set out to establish Ca(2+)-dependent signaling networks in ORN cilia by proteomic analysis. We subjected a ciliary membrane preparation to Ca(2+)/calmodulin-affinity chromatography using mild detergent conditions in order to maintain functional protein complexes involved in olfactory Ca(2+) signaling. Thus, calmodulin serves as a valuable tool to gain access to novel Ca(2+)-regulated protein complexes. Tandem mass spectrometry (nanoscale liquid-chromatography-electrospray injection) identified 123 distinct proteins. Ninety-seven proteins (79%) could be assigned to specific olfactory functions, including 32 to sensory signal transduction and 40 to cytoprotection. We point out novel perspectives for research on the Ca(2+)-signaling networks in the olfactory system of the rat.

Capecitabine and irinotecan as first-line chemotherapy in patients with metastatic colorectal cancer: results of an extended phase I study.

BACKGROUND: To define the maximum-tolerated dose (MTD) and to evaluate the dose-limiting toxicities (DLTs) of the combination of capecitabine and irinotecan in patients with metastatic colorectal cancer. PATIENTS AND METHODS: Thirty-seven patients with measurable metastatic colorectal cancer with no prior chemotherapy for metastatic disease were treated at three dose levels (DLs). For the first two dose levels, irinotecan (70 mg/m(2)) was administered once a week for 6 weeks in combination with 2 weeks of capecitabine at 1000 mg/m(2) (DL1) or 1250 mg/m(2) (DL2) twice daily, starting on days 1 and 22. In the last dose escalation step, the dose of irinotecan was increased to 80 mg/m(2) (DL3). One cycle lasted 7 weeks. RESULTS: In the subsequent phase I trial, 96 cycles of capecitabine and irinotecan were administered. At DL3, three out of six patients experienced DLTs (diarrhea, neutropenia, asthenia). In order to confirm the safety of the recommended dose, DL2 was extended to 15 patients. Five patients (33%) showed DLTs at this dose level, which was considered too high to embark on further clinical studies. Subsequently, the starting dose (DL1) was extended to a total of 16 patients, with diarrhea being the main toxicity. The overall response rate was 38% [95% confidence interval (CI) 21% to 58%], with a median response duration of 8.7 months (95% CI 6.4-11.5 months). CONCLUSIONS: The recommended doses for further studies are irinotecan 70 mg/m(2) and capecitabine 1000 mg/m(2). The combination of capecitabine and irinotecan appears to have significant therapeutic efficacy with manageable toxicity.

Facilitation of calmodulin-mediated odor adaptation by cAMP-gated channel subunits.

Calcium (Ca2+) influx through Ca2+-permeable ion channels plays a pivotal role in a variety of neuronal signaling processes, and negative-feedback control of this influx by Ca2+ itself is often equally important for modulation of such signaling. Negative modulation by Ca2+ through calmodulin (CaM) on cyclic nucleotide-gated (CNG) channels underlies the adaptation of olfactory receptor neurons to odorants. We show that this feedback requires two additional subunits of the native olfactory channel, CNGA4 and CNGB1b, even though the machinery for CaM binding and modulation is present in the principal subunit CNGA2. This provides a rationale for the presence of three distinct subunits in the native olfactory channel and underscores the subtle link between the molecular make-up of an ion channel and the physiological function it subserves.

Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression.

The suitability of the recently described red fluorescent protein dsRED from reef corals for use as a reporter in plant molecular biology was investigated. Based on the clone pDSRED (Clontech), plant expression vectors were constructed for constitutive dsRED expression in the cytosol, the endoplasmic reticulum and the vacuole. Fluorescence microscopy of tobacco BY2 suspension culture cells transiently expressing the plant vectors generated proved that cytosolic expression of the dsRED gives rise to readily detectable levels of red fluorescence, whereas expression in the ER was poor. Vacuolar dsRED expression did not result in any significant fluorescence. dsRED transgenic tobacco SR1 plants were generated to test the sensitivity of dsRED as a reporter in an autofluorescent background, and to identify the possible impact of the introduced fluorescent protein on morphogenesis, plant development and fertility. During the transformation and regeneration phase plants did not show any abnormalities, indicating that dsRED is not interfering with plant development and morphogenesis. Regenerated plants were analysed by PCR, Western blot and fluorescence microscopy for the presence and expression of the transferred genes. The filter sets chosen for fluorescence microscopy proved to be able to block the red chlorophyll fluorescence completely, allowing specific dsRED detection. Best expression levels were obtained with dsRED targeted to the cytosol or chloroplasts. ER-targeted expression of dsRED also gave rise to readily detectable fluorescence levels, whereas vacuolar expression yielded no fluorescence. dsRED transgenic plant lines expressing the protein in the cytosol, ER or chloroplast proved to be fertile. Seed set and germination were normal, except that the seeds and seedlings maintained the red fluorescence phenotype.

Oral capecitabine compared with intravenous fluorouracil plus leucovorin in patients with metastatic colorectal cancer: results of a large phase III study.

PURPOSE: To compare the efficacy and safety of orally administered capecitabine (Xeloda; Roche Laboratories, Inc, Nutley, NJ), a novel fluoropyrimidine carbamate designed to mimic continuous fluorouracil (5-FU) infusion but with preferential activation at the tumor site, with that of intravenous (IV) 5-FU plus leucovorin (5-FU/LV) as first-line treatment for metastatic colorectal cancer. PATIENTS AND METHODS: We prospectively randomized 602 patients to treatment with capecitabine 1,250 mg/m(2) administered twice daily days 1 to 14 every 3 weeks, or to the 4-weekly Mayo Clinic regimen (5-FU/LV) until disease progression or unacceptable toxicity. RESULTS: The primary objective, to demonstrate at least equivalent response rates in the two treatment groups, was met. The overall response rate was 18.9% for capecitabine and 15.0% for 5-FU/LV. In the capecitabine and 5-FU/LV groups, respectively, median time to disease progression was 5.2 and 4.7 months (log-rank P =.65); median time to treatment failure was 4.2 and 4.0 months (log-rank P =.89); and median overall survival was 13.2 and 12.1 months (log-rank P =.33). The toxicity profiles of both treatments were typical of fluoropyrimidines. However, capecitabine led to significantly lower incidences (P <.00001) of stomatitis and alopecia, but a higher incidence of cutaneous hand-foot syndrome (P <.00001). Capecitabine also resulted in lower incidences (P <.00001) of grade 3/4 stomatitis and neutropenia, leading to a lower incidence of grade 3/4 neutropenic fever and sepsis. Only grade 3 hand-foot syndrome (P <.00001) and uncomplicated grade 3/4 hyperbilirubinemia (P <.0001) were reported more frequently with capecitabine. CONCLUSION: Oral capecitabine achieved an at least equivalent efficacy compared with IV 5-FU/LV. Capecitabine demonstrated clinically meaningful safety advantages and the convenience of an oral agent.

[Cobra venom contains a protein belongs to the CRISP family]. / Iad kobry soderzhit belok CRISP-tipa.

Amino acid sequences of several fragments of the 25 k protein (molecular mass 24,953 Da) previously isolated from cobra Naja kaouthia (Kukhtina et al. Bioorg. Khim., 2000, vol. 26, pp. 803-807) were determined. Their comparison with the primary structures of known proteins showed that the 25 k protein belongs to the CRISP family and is the first protein of this type identified in cobra venoms.

Chemoelectrical signal transduction in olfactory sensory neurons of air-breathing vertebrates.

When odorants bind to the sensory cilia of olfactory sensory neurons, the cells respond with an electrical output signal, typically a short train of action potentials. This review describes the present state of knowledge about the olfactory signal transduction process. In the last decade, a set of transduction molecules has been identified which help to explain many aspects of the sensory response. Odor-induced second-messenger production, activation of transduction channels, the central role of the ciliary Ca2+ concentration, as well as mechanisms that mediate adaptation, are all qualitatively understood on the basis of a consistent scheme for chemoelectrical transduction. This scheme, although necessarily incomplete, can serve as a working model for further experimentation which may reveal kinetical aspects of signal transduction processes in olfactory sensory neurons.

Fraction of the dark current carried by Ca(2+) through cGMP-gated ion channels of intact rod and cone photoreceptors.

The selectivity for Ca(2+) over Na(+), PCa/PNa, is higher in cGMP-gated (CNG) ion channels of retinal cone photoreceptors than in those of rods. To ascertain the physiological significance of this fact, we determined the fraction of the cyclic nucleotide-gated current specifically carried by Ca(2+) in intact rods and cones. We activated CNG channels by suddenly (<5 ms) increasing free 8Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide-dependent changes in membrane current and fluorescence of the Ca(2+)-binding dye, Fura-2, also loaded into the cells. The ratio of changes in fura-2 fluorescence and the integral of the membrane current, under a restricted set of experimental conditions, is a direct measure of the fractional Ca(2+) flux. Under normal physiological salt concentrations, the fractional Ca(2+) flux is higher in CNG channels of cones than in those of rods, but it differs little among cones (or rods) of different species. Under normal physiological conditions and for membrane currents

Permeation and activation of the M2 ion channel of influenza A virus.

The M(2) ion channel protein of influenza A virus is essential for mediating protein-protein dissociation during the virus uncoating process that occurs when the virus is in the acidic environment of the lumen of the secondary endosome. The difficulty of determining the ion selectivity of this minimalistic ion channel is due in part to the fact that the channel activity is so great that it causes local acidification in the expressing cells and a consequent alteration of reversal voltage, V(rev). We have confirmed the high proton selectivity of the channel (1.5-2.0 x 10(6)) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of V(rev) with proton equilibrium potential; 2) measurement of pH(in) and V(rev) while Na(+)(out) was replaced; 3) measurements with limiting external buffer concentration to limit proton currents specifically; and 4) comparison of measurements of M(2)-expressing cells with cells exposed to a protonophore. Increased currents at low pH(out) are due to true activation and not merely increased [H(+)](out) because increased pH(out) stops the outward current of acidified cells. Although the proton conductance is the biologically relevant conductance in an influenza virus-infected cell, experiments employing methods 1-3 show that the channel is also capable of conducting NH(4)(+), probably by a different mechanism from H(+).

Neuronal Ca2+ -activated Cl- channels--homing in on an elusive channel species.

Ca2+ -activated Cl- channels control electrical excitability in various peripheral and central populations of neurons. Ca2+ influx through voltage-gated or ligand-operated channels, as well as Ca2+ release from intracellular stores, have been shown to induce substantial Cl- conductances that determine the response to synaptic input, spike rate, and the receptor current of various kinds of neurons. In some neurons, Ca2+ -activated Cl- channels are localized in the dendritic membrane, and their contribution to signal processing depends on the local Cl- equilibrium potential which may differ considerably from those at the membranes of somata and axons. In olfactory sensory neurons, the channels are expressed in ciliary processes of dendritic endings where they serve to amplify the odor-induced receptor current. Recent biophysical studies of signal transduction in olfactory sensory neurons have yielded some insight into the functional properties of Ca2+ -activated Cl- channels expressed in the chemosensory membrane of these cells. Ion selectivity, channel conductance, and Ca2+ sensitivity have been investigated, and the role of the channels in the generation of receptor currents is well understood. However, further investigation of neuronal Ca2+ -activated Cl- channels will require information about the molecular structure of the channel protein, the regulation of channel activity by cellular signaling pathways, as well as the distribution of channels in different compartments of the neuron. To understand the physiological role of these channels it is also important to know the Cl- equilibrium potential in cells or in distinct cell compartments that express Ca2+ -activated Cl- channels. The state of knowledge about most of these aspects is considerably more advanced in non-neuronal cells, in particular in epithelia and smooth muscle. This review, therefore, collects results both from neuronal and from non-neuronal cells with the intent of facilitating research into Ca2+ -activated Cl- channels and their physiological functions in neurons.

The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits.

Cyclic nucleotide-gated (CNG) channels play central roles in visual and olfactory signal transduction. In the retina, rod photoreceptors express the subunits CNCalpha1 and CNCbeta1a. In cone photoreceptors, only CNCalpha2 expression has been demonstrated so far. Rat olfactory sensory neurons (OSNs) express two homologous subunits, here designated CNCalpha3 and CNCalpha4. This paper describes the characterization of CNCbeta1b, a third subunit expressed in OSNs and establishes it as a component of the native channel. CNCbeta1b is an alternate splice form of the rod photoreceptor CNCbeta1a subunit. Analysis of mRNA and protein expression together suggest co-expression of all three subunits in sensory cilia of OSNs. From single-channel analyses of native rat olfactory channels and of channels expressed heterologously from all possible combinations of the CNCalpha3, -alpha4, and -beta1b subunits, we conclude that the native CNG channel in OSNs is composed of all three subunits. Thus, CNG channels in both rod photoreceptors and olfactory sensory neurons result from coassembly of specific alpha subunits with various forms of an alternatively spliced beta subunit.

Ca2+ permeation in cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non-excitable cells, co-assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+-binding affinity of the intrapore cation-binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.

Synthesis, photochemistry and application of (7-methoxycoumarin-4-yl)methyl-caged 8-bromoadenosine cyclic 3',5'-monophosphate and 8-bromoguanosine cyclic 3',5'-monophosphate photolyzed in the nanosecond time region.

New caged derivatives of hydrolysis-resistant 8-bromoadenosine cyclic 3',5'-monophosphate (8-Br-cAMP) and 8-bromoguanosine cyclic 3',5'-monophosphate (8-Br-cGMP) are described. The compounds are the axial and equatorial isomers of the (7-methoxycoumarin-4-yl)methyl (MCM) esters of cyclic nucleotides. Synthesis is accomplished by treatment of 4-bromomethyl-7-methoxycoumarin with the tetra-n-butylammonium salts of the 8-bromo-substituted cyclic nucleotides or with the free acids of 8-Br-cAMP and 8-Br-cGMP in the presence of silver(I) oxide. MCM-caged 8-Br-cAMP and MCM-caged 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light within a few nanoseconds. They show favorable absorption properties and quantum yields and are resistant to hydrolysis in aqueous buffer solutions. The moderate fluorescence properties of the caged compounds in comparison with the strongly fluorescent 4-hydroxymethyl-7-methoxycoumarin (MCM-OH) photoproduct allow the indirect estimation of the amount of photolytically released cyclic nucleotides in aqueous buffer solutions using fluorescence measurements. Their usefulness for physiological studies has been examined in a mammalian cell line expressing the cyclic nucleotide-gated ion channel of bovine olfactory sensory neurons using the patch-clamp technique and confocal laser scanning microscopy. The caged compounds serve as efficient and rapid intracellular sources of 8-Br-cAMP and 8-Br-cGMP. However, at least in HEK 293 cells, fluorescence signals cannot be used to monitor the photolysis of MCM-caged 8-Br-cAMP and 8-Br-cGMP, due to quenching of the fluorescence of MCM-OH.

A depolarizing chloride current contributes to chemoelectrical transduction in olfactory sensory neurons in situ.

Recent biophysical investigations of vertebrate olfactory signal transduction have revealed that Ca2+-gated Cl- channels are activated during odorant detection in the chemosensory membrane of olfactory sensory neurons (OSNs). To understand the role of these channels in chemoelectrical signal transduction, it is necessary to know the Cl--equilibrium potential that determines direction and size of Cl- fluxes across the chemosensory membrane. We have measured Cl-, Na+, and K+ concentrations in ultrathin cryosections of rat olfactory epithelium, as well as relative element contents in isolated microsamples of olfactory mucus, using energy-dispersive x-ray microanalysis. Determination of the Cl- concentrations in dendritic knobs and olfactory mucus yielded an estimate of the Cl--equilibrium potential ECl in situ. With Cl- concentrations of 69 mM in dendritic knobs and 55 mM in olfactory mucus, we obtained an ECl value of +6 +/- 12 mV. This indicates that Ca2+-gated Cl- channels in olfactory cilia conduct inward currents in vivo carried by Cl- efflux into the mucus. Our results show that rat OSNs are among the few known types of neurons that maintain an elevated level of cytosolic Cl-. In these cells, activation of Cl- channels leads to depolarization of the membrane voltage and can induce electrical excitation. The depolarizing Cl- current in mammalian OSNs appears to contribute a major fraction to the receptor current and may sustain olfactory function in sweet-water animals.

Characterization of ether-à-go-go channels present in photoreceptors reveals similarity to IKx, a K+ current in rod inner segments.

In this study, we describe two splice variants of an ether-à-go-go (EAG) K+ channel cloned from bovine retina: bEAG1 and bEAG2. The bEAG2 polypeptide contains an additional insertion of 27 amino acids in the extracellular linker between transmembrane segments S3 and S4. The heterologously expressed splice variants differ in their activation kinetics and are differently modulated by extracellular Mg2+. Cooperativity of modulation by Mg2+ suggests that each subunit of the putative tetrameric channel binds a Mg2+ ion. The channels are neither permeable to Ca2+ ions nor modulated by cyclic nucleotides. In situ hybridization localizes channel transcripts to photoreceptors and retinal ganglion cells. Comparison of EAG currents with IKx, a noninactivating K+ current in the inner segment of rod photoreceptors, reveals an intriguing similarity, suggesting that EAG polypeptides are involved in the formation of Kx channels.