First-line therapy with moderate dose capecitabine in metastatic breast cancer is safe and active: results of the MONICA trial.

BACKGROUND: To determine activity and safety of capecitabine at a moderate dose of 2000 mg/m(2) as first-line therapy for metastatic breast cancer. METHODS: In this prospective phase II trial, patients with HER2-negative metastatic breast cancer received first-line capecitabine 2000 mg/m(2) on days 1-14 every 3 weeks. The primary aim was to exclude a time to progression (TTP) <6 months. Secondary end-points were overall response rate, overall survival (OS), toxicity and quality of life. RESULTS: Median age of the 161 included patients was 65 years. Median TTP and OS were 7.3 months [95% (confidence interval) CI: 6.2-8.4] and 17.1 months (95% CI: 14.0-20.3), respectively. An overall response rate of 26.1%, including 13 complete remissions was observed. Patients developing grade I-III hand-foot syndrome had a significantly longer TTP and OS and patients >65 years also achieved a significantly longer TTP. Haematological grade I-IV toxicities were leucopenia (64.0%), anaemia (50.9%) and thrombocytopenia (28.0%). Relevant non-haematological toxicities were hand-food-syndrome (37.3%), fatigue (34.2%), nausea (29.8%) and diarrhoea (20.5%). Quality of life assessment revealed an improved emotional function, but worsening of nausea and vomiting from cycle 1-10. CONCLUSIONS: Capecitabine at a dose of 2000 mg/m(2) is active and safe as first-line treatment of patients with metastatic breast cancer.

A new sarcomatoid variant of multiple myeloma in a 20-year-old male.

We present a 20-year-old man with a rare variant of multiple myeloma with peculiar spindle cell morphology and sarcomatoid growth. The diagnosis of multiple myeloma was substantiated by clinical examinations. The patient was treated with several therapeutic trials including autologous stem cell support. Unfortunately, he developed a disseminated aspergillosis of the lungs and died of fatal lung bleeding. We recommended that "sarcomatous" multiple myeloma be considered in cases of "unclassifiable" sarcomatous tumors of the bone marrow.

Anonymous predictive testing for Huntington's disease in the United States.

The widespread use of a predictive genetic test for Huntington's disease (HD) since 1993 has brought to the forefront issues regarding genetic privacy. Although the possibility of anonymous genetic testing has been discussed, its use in the United States has not been described previously. We review the experiences of 11 genetics specialists with anonymous predictive testing for HD. We found that more men than women requested anonymous testing, for reasons that more often related to personal privacy than to insurance or discrimination concerns. A number of approaches to anonymity were used, and genetics specialists varied in the degree to which they were comfortable with the process. A number of legal, medical, and practical questions are raised, which will require resolution if anonymous testing is to be performed with a greater frequency in the future.

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

Measurement of intracellular Ca2+ changes using novel caged cyclic nucleotides and confocal laser scanning microscopy.

The intention of this study is to explore the applicability of confocal microscopy in conjunction with the use of caged cyclic nucleotide derivatives. The methodological potential of UV laser confocal microscopy has been assessed. It is shown that illumination of a single cell or a small area of a single cell is possible, whereby the intracelluar Ca2+ signal is measured at illuminated and non-illuminated cells. Such measurements do not have a high time resolution because of the specific system parameters. However, with an N2 pulse laser (not part of the standard microscope set-up), Ca2+ signals with a time resolution of around 100 ms have been measured. This facilitates investigation of the kinetics of Ca2+ influx. Intracellular Ca2+ measurements at HEK293 and sperm cells have been made here. For sperm cells the advantages of confocal microscopy are best evidenced in conjunction with the use of caged cyclic nucleotides; a cyclic nucleotide-gated Ca2+ influx at the tail of these cells has thereby been demonstrated for the first time.

Analysis of a very large trinucleotide repeat in a patient with juvenile Huntington's disease.

A patient with juvenile Huntington's disease (HD) of probable maternal inheritance is reported. The expanded IT-15 allele was only detected with the use of modified PCR and Southern transfer techniques, which showed a CAG trinucleotide repeat expansion of approximately 250 repeats-the largest CAG expansion reported within the huntingtin gene. This case emphasizes the need for communication between the diagnostic laboratory and the clinician to define the molecular genetics of unusual cases.

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.

Cyclic nucleotide-gated channels on the flagellum control Ca2+ entry into sperm.

Cyclic nucleotide-gated (CNG) channels are key elements of cGMP- and cAMP-signaling pathways in vertebrate photoreceptor cells and in olfactory sensory neurons, respectively. These channels form heterooligomeric complexes composed of at least two distinct subunits (alpha and beta). The alpha subunit of cone photoreceptors is also present in mammalian sperm. Here we identify one short and several long less abundant transcripts of beta subunits in testis. The alpha and beta subunits are expressed in a characteristic temporal and spatial pattern in sperm and precursor cells. In mature sperm, the alpha subunit is observed along the entire flagellum, whereas the short beta subunit is restricted to the principal piece of the flagellum. These findings suggest that different forms of CNG channels coexist in the flagellum. Confocal microscopy in conjunction with the Ca2+ indicator Fluo-3 shows that the CNG channels serve as a Ca2+ entry pathway that responds more sensitively to cGMP than to cAMP. Assuming that CNG channel subtypes differ in their Ca2+ permeability, dissimilar localization of alpha and beta subunits may give rise to a pattern of Ca2+ microdomains along the flagellum, thereby providing the structural basis for control of flagellar bending waves.

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.

Novel caged compounds of hydrolysis-resistant 8-Br-cAMP and 8-Br-cGMP: photolabile NPE esters.

The application of the 1-(2-nitrophenyl)ethyl (NPE) moiety as a photolabile ligand for the release of hydrolysis-resistant 8-Br-cAMP and 8-Br-cGMP was examined. NPE-caged 8-Br-cAMP and 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light. The synthesis procedure resulted in diastereoisomeric mixtures, which were chromatographically separated into the axial and equatorial isomers of NPE-caged 8-Br-cAMP and 8-Br-cGMP. The hydrolytic stability, solubility and photochemical properties of these derivatives were compared to the previously reported 4.5-dimethoxy-2-nitrobenzyl (DMNB) compounds. We found that the axial isomers of NPE-caged 8-Br-cAMP and 8-Br-cGMP had a considerably better solvolytic stability than the respective equatorial isomers as well as the DMNB-caged derivatives. Their usefulness for physiological studies was examined in a mammalian cell line expressing the cyclic nucleotide-gated (CNG) ion channel of bovine olfactory sensory neurons.

The Na/Ca-K exchanger of rod photoreceptor exists as dimer in the plasma membrane.

The oligomeric state of the Na/Ca-K exchanger in the plasma membrane of bovine photoreceptors was investigated using chemical cross-linking techniques. In the natural membrane, virtually all Na/Ca-K exchanger could be cross-linked mainly to a complex having an apparent molecular mass of 490 kDa by cupric phenanthroline catalyzed disulfide bonding as evidenced by Western blotting. Stable cross-links of the exchanger were also obtained with the thiol-specific reagent N,N'-p-phenylidenedimaleimide. Neuraminidase treatment reduced the apparent molecular mass of the highly glycosylated Na/Ca-K exchanger and of the 490 kDa cross-link product by 50 and 85 kDa, respectively. DL-1,4-Bismaleimido-2,3-butanediol (BMBD), a novel cleavable dimaleimide, was synthesized in order to produce cross-links that were stable to reductive conditions. Purification of the BMBD cross-linked exchanger followed by two-dimensional SDS polyacrylamide electrophoresis identified the cross-linked homodimers of the exchanger. There was no indication of higher oligomers, suggesting that the exchanger exists as a dimer in the plasma membrane. Hydrodynamic properties of the detergent-solubilized exchanger were determined by velocity sedimentation and gel filtration chromatography. The Triton X-100-solubilized exchanger ran as a single species having a Stokes radius of 10.0 nm, a sedimentation coefficient of 5.4 S, and a partial specific volume of 0.74 mL/g in Triton X-100. Similar results were obtained for the CHAPS-solubilized exchanger. A molecular mass of 236 and 205 kDa was calculated for the exchanger-detergent complex and the detergent-free protein, respectively. Neuraminidase treatment further reduced the molecular mass of the exchanger indicating that glycosylation contributes significantly to the mass of the exchanger. Cross-links of the exchanger were not detected if cross-linking was attempted after solubilization in 10 mM CHAPS. However, after reconstitution of the purified exchanger into soybean phosphatidylcholine vesicles, chemical cross-linking yielded again dimers. On the basis of these cross-linking and hydrodynamic studies, we conclude that the exchanger exists as a homodimer in the rod outer segment plasma membrane but dissociates into a monomer when solubilized in detergent.

Caged compounds of hydrolysis-resistant analogues of cAMP and cGMP: synthesis and application to cyclic nucleotide-gated channels.

Photolabile compounds which rapidly release cAMP or cGMP after photolysis are widely used for in situ studies of signaling pathways inside cells. We synthesized two novel caged compounds, 4,5-dimethoxy-2-nitrobenzyl 8-Br-cAMP (caged 8-Br-cAMP) and 4,5-dimethoxy-2-nitrobenzyl 8-Br-cGMP caged 8-BR-cGMP), which respectively release the hydrolysis-resistant analogues 8-Br-cAMP and 8-Br-cGMP. Their usefulness for physiological studies was examined in a mammalian cell line expressing the cyclic nucleotide-gated (CNG) ion channel of bovine olfactory sensory neurons. The synthesis procedure resulted in diastereomeric mixtures which were chromatographically separated into the axial and equatorial isomers of caged 8-BR-cAMP and of caged 8-BR-cGMP. The axial isomers which have a higher solubility and better solvolytic stability than the equatorial forms were used for experiments with CNG channels. Flashes of UV light produced steps in the concentration of 8-Br-cGMP which activated currents through CNG channels. Concentration steps inside the cell could be calibrated precisely using the relation between the ligand concentration and the normalized current. Similar results were obtained with caged 8-Br-cAMP. Control experiments with caged cGMP showed that flash-induced currents decayed within a few minutes because photoreleased cGMP was degraded by endogenous phosphodiesterase activity. The rise time of the 8-Br-cGMP-activated whole-cell current was consistent with a bimolecular reaction between channel and ligand.

[Pharmacokinetics and biotransformation of 3-cyan-2-morpholino-5-(pyrid-4-yl)pyridine (AWD 122-14) in the rat]. / Pharmakokinetik und Biotransformation von 3-Cyan-2-morpholino-5-(pyrid-4-yl)pyridin (AWD 122-14) an der Ratte.

A sensitive isocratic HPLC method for the analysis of AWD 122-14 (1) in plasma and urine was developed. The extraction was processed on-line on a short column. The pharmacokinetics of 1 was studied in rats. The plasma concentration-time course was described by an 1-compartment-model. The pharmacokinetic parameters were estimated. The absorption and elimination of 1 are relatively fast. A single oral dose (D = 1 x 10(-5) mol/kg) was rapidly absorbed (absorption rate constant: kI = 6.85 h-1). The elimination rate constant is kE = 1.59 h-1. The absolute bioavailability of a single oral dose equals 11%. In rats 1 is mainly metabolized. Less than 5% of the dose is excreted in the urine as unchanged drug. The chemical structures of 10 of the 12 isolated metabolites were determined using high-resolution mass spectrometry.

Solid-phase synthesis and stability of triple-helical peptides incorporating native collagen sequences.

A generally applicable solid-phase methodology has been developed for the synthesis of triple-helical polypeptides incorporating native collagen sequences. Three nascent peptide chains are C-terminal linked through one N alpha-amino and two N epsilon-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. Different protecting group strategies, including several three-dimensionally orthogonal schemes, have been utilized for the synthesis of four homotrimeric triple-helical polypeptides (THPs) of 79-124 residues, three of which incorporate native type IV collagen sequences. Highly efficient assemblies were achieved by 9-fluorenylmethoxycarbonyl (Fmoc) N alpha-amino group protection, in situ 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate mediated couplings, and 1,8-diazabicyclo [5.4.0] undec-7-ene mediated Fmoc group removal. THPs were characterized by Edman degradation sequencing, size-exclusion chromatography, mass spectrometry, reversed-phase high performance liquid chromatography, and CD spectroscopy. THP thermal stabilities ranged from 35 to 59 degrees C, with chain length and Hyp content being the influential factors. Melting temperatures and van't Hoff enthalpies for peptide triple-helical denaturation could be correlated well to Hyp content. The THP synthetic protocol developed here will allow for the study of both structure and biological activity of specific collagen sequences in homotrimeric and heterotrimeric forms.

[Potential cardiotonics. 16. Molecular and crystal structures of three polymorphic forms and a hydrochloride monohydrate of 3-cyano-2-morpholino-5-(pyridin-4-yl)pyridine (AWD 122-14)]. / Potentielle Kardiotonika. 16. Mitteilung3: Molekül-und Kristallstrukturen dreier polymorpher Formen und eines Hydrochlorid-Monohydrates von 3-Cyan-2-morpholino-5-(pyrid-4-yl) pyridin (AWD 122-14).

The results of an X-ray structure analysis of the alpha-modification were the starting point for the prediction and establishment of further polymorphic and pseudopolymorphic, respectively, forms of AWD 122-14. A complete structure determination of the gamma- and delta-modifikation as well as of a hydrochloride monohydrate could be carried out. Molecular parameters, conformational flexibility, and intermolecular interactions are discussed in this paper.