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.

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

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.

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.

Effects of gossypol on the cell cycle phases in T-47D human breast cancer cells.

Since gossypol, a naturally occurring component of cottonseed oil, exhibits a broad spectrum of activities, we have examined it as an antitumor agent on breast cancer. The effects of different concentrations of gossypol on the T-47D human breast cancer cell cycle phases were studied using cytometric image processing on Feulgen stained nuclei. The proportion of cells at different cell cycle phases was determined by discriminate analysis of the image parameters and gave good classification ranging from 86 to 100%. Gossypol was found to increase the G0/G1 fraction of the T-47D cells. This cell kinetic alteration by gossypol was shown to be dose dependent and reversible. Complete reversal of the effect of gossypol was observed after four days with a simple change to gossypol-free medium. The cells then progressed into S and G2/M phase, thus indicating that gossypol-treated cells remain viable. Gossypol was shown to have a strong inhibitory effect on cellular proliferation in T-47D cells. It was also found that this agent is only toxic to cells at the highest dose tested (10 microM). The results of this study may be of clinical significance in the treatment of breast cancer, since gossypol shows strong antiproliferative properties.

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.

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.

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.

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.

[Hansch analysis of the inhibitory action of 3- and 4-substituted benzamidines on thrombin, plasmin and trypsin (author's transl)]. / Hansch-Analyse der Hemwirkung von 3- und 4-substituierten Benzamidinen gegenüber Thrombin, Plasmin und Trypsin.

Using Hansch formalism, the suthors studied quantitatively the relationship between the structure and the inhibitory action of 3- and 4-substituted amidinophenyl derivatives on thrombin, plasmin and trypsin. It was found that the inhibitory action on all three enzymes depends in the same way from the hydrophobic and electronic properties of the substituents and from an additional term in case of substituents with X-CO-Y structure. The predictive value of the equations is satisfactory.

[Free-Wilson analysis of the inhibitory effect of 4-substituted benzamidines on thrombin, plasmin and trypsin (author's transl)]. / Free-Wilson-Analyse der Hemmwirkung von 4-substituierten Benzamidinen gegenüber Thrombin, Plasmin und Trypsin.

Using the Fujita-Ban model, the authors studied the quantitative structure-inhibitory activity relationships of a series of 4-amidinophenyl compounds with regard to thrombin, plasmin and trypsin. A satisfactory specification of the inhibitory activities was obtained from activity increments of molecular segments.

[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.

[Potential cardiotonic agents. 10. Synthesis and positive inotropic action of 5-(4-pyridinyl)- and 5-phenyl-substituted 2-alkylthio-3-cyan-pyridines and their S-oxidation products]. / Potentielle Kardiotonika. 10. Mitteilung3: Darstellung und positiv inotrope Wirkung von 5-(pyrid-4-yl)-und 5-phenyl-substituierten 2-Alkylthio-3-cyanpyridinen und ihren S-Oxidationsprodukten.

The alkylation of the 5-(4-pyridinyl)- and 5-phenyl-substituted 3-cyan-2(1H)-pyridinethiones 1 with alkyl iodides and halomethylcarbonyl compounds led to the 2-alkylthio-pyridines 3a-c, 3e, 3f and the thieno[2,3-b]pyridines 4, respectively. In an other way the 2-ethylthio-5-(4-pyridinyl)pyridines 3b and 3d were formed from the corresponding 2-chloroderivatives and ethylmercaptan. By means of oxidation of the 2-alkylthio-pyridines 3 with 3-chloroperbenzoic acid and potassium permanganate, respectively, the 2-sulfinyl- and 2-sulfonyl-pyridines 5 and 6 were obtained. The 3-cyano-5-(4-pyridinyl)pyridine-2-sulfonic acid 7 was prepared by oxidation of the 2(1H)-pyridinethione 1a with potassium permanganate. Some derivatives possess positive inotropic properties.

[Potential cardiotonics. 13. Crystal structure, charge distribution and molecular electrostatic potential of 3-cyano-2-(3-diethyl-aminopropylamino)-5-(4-pyridinyl)pyridine (AWD 122-60)]. / Potentielle Kardiotonika. 13. Mitteilung: Kristallstruktur, Ladungsverteilung und molekulares elektrostatisches Potential von 3-Cyan-2-(3-diethylamino-propylamino)-5-(pyrid-4-yl)pyridin (AWD 122-60).

The crystal and molecular structure of the potential cardiotonic agent AWD 122-60 have been determined by X-ray structure analysis. Based on the molecular structure charge distribution and molecular electrostatic potential were evaluated. The results are discussed in comparison with those of other cardiotonic 3,4'-bipyridines.

[Quantum chemical studies on substituted benzamidines as inhibitors of the serine proteinases trypsin and thrombin]. / Quantenchemische Untersuchungen an substituierten Benzamidinen als Inhibitoren der Serinproteinasen Trypsin und Thrombin.

Substituted benzamidines are competitive inhibitors of trypsin and thrombin. The aim of this work is to find an interpretation for the relation between chemical structure of the inhibitors and the activity on the both enzymes by quantum chemical methods. For the description of the electrostatic interaction between inhibitor and enzyme the molecular electrostatic potential (MEP) was used. The interpretation took place with the help of a simple model of reactive groups in the enzyme. The obtained results were discussed and compared with those resulting from CNDO/2--reactivity indices. The application of the MEP shows its advantage to investigations of enzyme-inhibitor interactions.

[The antianaphylactic effect of derivatives of 4-oxoquinazoline, 4-oxoquinazol-3-yl-benzoic acid and 4-oxoquinazol-3-yl-acetic acid in the rat]. / Untersuchungen zur antianaphylaktischen Wirksamkeit von Derivaten des 4-Oxochinazolins, der 4-Oxochinazolin-3-yl-benzoesäure und der 4-Oxochinazolin-3-yl-essigsäure an der Ratte.

Derivatives substituted at position 2 of 4-oxochinazolin as well as derivates of oxochinazolin-3-yl-benzoic acid and 4-oxochinazolin-3-yl-acetic acid had been tested for their antianaphylactic activities in the passive (PCA) and/or active cutaneous anaphylaxis (ACA) in rats. At i.p. administration (ACA) or intracutaneous (i.c.) administration (PCA) most of the derivates displayed a moderate antianaphylactic activity which et best could be compared to the theophylline activity. Only the free acids of the 4-oxochinazolin-3-yl-acetic acid derivatives were active at i.c. administration. All compounds tested remained inactive at p.o. administration.

[X-ray structural analysis of 3 crystalline modifications of 3-cyan-5-methyl-5-(pyridinyl)-1,2-dihydro-pyrid-2-one (milrinone)]. / Röntgenstrukturanalytische Untersuchungen an drei kristallinen Modifikationen des 3-Cyan-6-methyl-5-(pyrid-4-yl)-1,2-dihydropyrid-2-on (Milrinon).

The crystal and molecular structures of the alpha-, beta-, and gamma-modification of the cardiotonic compound milrinone have been determined by X-ray structure analyses. In all modifications the molecules exist in the lactam form. Because of steric hindrances the pyridine rings deviate considerably from coplanarity. The dihedral angle amounts to 45 degrees in milrinone-alpha, 43.7 degrees in milrinone-beta, and 57.5 degrees in milrinone-gamma. In the three crystal structures molecules are connected via hydrogen bonds forming dimers. These are centrosymmetric in milrinone-alpha and -beta. In milrinone-gamma the molecules within a dimer are symmetry related by a twofold axis. The dimers are linked by charge transfer interactions. This leads to infinite chains in the alpha- and beta-modification and to infinite layers in milrinone-gamma.

[Potential cardiotonics. 14. Synthesis and in vitro positive inotropic actions of 4-morpholino-5-(4-pyridinyl)pyridine derivatives]. / Potentielle Kardiotonika. 14. Mitteilung: Synthese und positiv inotrope Wirkungen in vitro von 2-Morpholino-5-(pyrid-4-yl)pyridin-Derivaten.

By the reaction of 2-chloro-5-(4-pyridinyl)pyridines 1-6 with morpholine as well as by derivation of the 2-morpholino-pyridine-3-carboxamide 8 the 3-substituted 2-morpholino-5-(4-pyridinyl)pyridines 7-14 were prepared. The evaluation for positive inotropic properties in spontaneously beating isolated guinea pig atria gave for the 3-cyano derivative 7 (AWD 122-14) the best activity. The potency is comparable to that of milrinone and is due to partial by inhibition of phosphodiesterase III (PDE III).

[Potential cardiotonics. 15. Crystal modifications of 3-cyano-2-morpholino-5-(4-pyridinyl)pyridine (AWD 122-14)]. / Potentielle Kardiotonika. 15. Mitteilung: Kristallmodifikationen von 3-Cyan-2-morpholino-5-(pyrid-4-yl)pyridin (AWD 122-14).

The preparation and X-ray crystallographic characterisation of 10 polymorphic or pseudo-polymorphic forms of the novel cardiotonic AWD 122-14 are described. Thermic transformations of some polymorphic forms into the alpha-form was proved by thermogravimetric analysis and powder diffraction pattern. In dissolution behaviour no significant differences were found between the crystalline modifications but with regard to galenic processing compact alpha-, gamma- and epsilon-forms show advantages.

[Potential cardiotonic agents. 1. Synthesis and pharmacologic properties of 3-cyano-2-oxaalkylamino-5-(4-pyridinyl)pyridines]. / Potentielle Kardiotonika. 1. Mitteilung: Darstellung und pharmakologische Eigenschaften von 3-Cyan-2-oxaalkylamino-5-(pyrid-4-yl)pyriden.

Synthesis, physicochemical properties and evaluation of positive inotropic and vasodilator activities are described in a series of substituted 2-amino-3-cyano-5-(4-pyridinyl)pyridines. Some of the 2-oxaalkylamino derivatives showed remarkable positive inotropic activities and, additionally, a decrease in blood pressure. The most potent compounds 11 and 13 have a greater activity than amrinone.