Single step isolation of sildenafil from commercially available Viagra tablets.

Sildenafil, the active ingredient in Viagra, has been purified from commercially available tablets. The purification, using Sephadex G25 chromatography under conditions of low ionic strength, is simple and inexpensive. Sildenafil purified according to this protocol has been characterized with respect to its IC50 for PDE5, its ultraviolet absorption profile, and by collision-induced dissociation fingerprinting, positive ion nanospray, and MALDI mass spectrometry.

Plasma steroid and nutrient levels during the active season in wild Testudo horsfieldi.

Plasma concentrations of sex steroids (testosterone and progesterone), proteins (total protein and albumin), lipids (phospholipids, cholesterol, and triglycerides), and minerals (calcium and phosphorus) were measured in wild Testudo horsfieldi in Uzbekistan, during the short, 3-month activity period (March-May, 1998). Testosterone concentration in males was highest (52 ng/ml) when they had just emerged from brumation (hibernation) in mid-March, which was also the beginning of the mating period, and fell in April. In females, progesterone peaked in mid-April (at 10 ng/ml), just before ovulation of the first clutches at the end of April and beginning of May.

Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches.

To directly test the requirement for hedgehog signaling in the telencephalon from early neurogenesis, we examined conditional null alleles of both the Sonic hedgehog and Smoothened genes. While the removal of Shh signaling in these animals resulted in only minor patterning abnormalities, the number of neural progenitors in both the postnatal subventricular zone and hippocampus was dramatically reduced. In the subventricular zone, this was partially attributable to a marked increase in programmed cell death.

Dimerization of cGMP-dependent protein kinase Ibeta is mediated by an extensive amino-terminal leucine zipper motif, and dimerization modulates enzyme function.

All mammalian cGMP-dependent protein kinases (PKGs) are dimeric. Dimerization of PKGs involves sequences located near the amino termini, which contain a conserved, extended leucine zipper motif. In PKG Ibeta this includes eight Leu/Ile heptad repeats, and in the present study, deletion and site-directed mutagenesis have been used to systematically delete these repeats or substitute individual Leu/Ile.

Combinatorial function of the homeodomain proteins Nkx2.1 and Gsh2 in ventral telencephalic patterning.

Regional patterning of the mammalian telencephalon requires the function of three homeodomain-containing transcription factors, Pax6, Gsh2 and Nkx2.1. These factors are required for the development of the dorsal, lateral and medial domains of the telencephalon, respectively.

Dlx2 progenitor migration in wild type and Nkx2.1 mutant telencephalon.

The transcription factor Dlx2 is expressed widely throughout the ventral telencephalon. We have examined the in vitro and in vivo migration of Dlx2 progenitors originating from the different ganglionic eminences of both wild type and Nkx2.1 mutant animals.

[3H]sildenafil binding to phosphodiesterase-5 is specific, kinetically heterogeneous, and stimulated by cGMP.

Sildenafil (Viagra) potentiates penile erection by acting as a nonhydrolyzable analog of cGMP and competing with this nucleotide for catalysis by phosphodiesterase-5 (PDE5), but the characteristics of direct binding of radiolabeled sildenafil to PDE5 have not been determined. [3H]Sildenafil binding to PDE5 was retained when filtered through nitrocellulose or glass-fiber membranes. Binding was inhibited by excess sildenafil, 2-(2-methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), 3-isobutyl-1-methylxanthine, EDTA, or cGMP, but not by cAMP or 5'-GMP.

The body in health and illness.

In this keynote address, the author discusses perception of the body in the context of chronic illness compared with that of health. She describes changes that occur in illness with respect to time, space, morality, aesthetics, morality, technology, information, and interpersonal relationships using examples from her research, and explores the construction of illness and health identities.

cGMP-dependent protein kinase protects cGMP from hydrolysis by phosphodiesterase-5.

The physiological effects of cGMP are largely determined by the activities of intracellular receptors, including cGMP-dependent protein kinase (PKG) and cGMP-binding cyclic nucleotide phosphodiesterases (PDEs), and the distribution of cGMP among these receptors dictates activity of the signalling pathway. In the present study, the effects of PKG-Ialpha or PKG-Ibeta on the rate of cGMP hydrolysis by the isolated PDE5 catalytic domain were examined. PKG-Ialpha strongly inhibited cGMP hydrolysis with an IC(50) value of 217 nM, which is similar to the physiological concentration of PKG in pig coronary artery reported previously.

Mechanisms associated with cGMP binding and activation of cGMP-dependent protein kinase.

Using small-angle x-ray scattering, we have observed the cGMP-induced elongation of an active, cGMP-dependent, monomeric deletion mutant of cGMP-dependent protein kinase (Delta(1-52)PKG-I beta). On saturation with cGMP, the radius of gyration of Delta(1-52)PKG-I beta increases from 29.4 +/- 0.

Suppressor of cytokine signaling-1 has IFN-gamma-independent actions in T cell homeostasis.

Suppressor of cytokine signaling (SOCS)-1 is a member of a family of proteins that negatively regulate cytokine signaling pathways. We have previously established that SOCS-1 is a key regulator of IFN-gamma signaling and that IFN-gamma is responsible for the complex inflammatory disease that leads to the death of SOCS-1-deficient mice. In this study, we provide evidence that SOCS-1 is also a critical regulator of IFN-gamma-independent immunoregulatory factors.

Cytoskeletal disrupting agents prevent calmodulin kinase, IQ domain and voltage-dependent facilitation of L-type Ca2+ channels.

A calmodulin (CaM) binding 'IQ' domain on the L-type Ca(2+) channel (LTCC) C terminus and calmodulin kinase II (CaMK) both signal increases in LTCC opening probability (P(o)) by shifting LTCCs into a gating mode (mode 2) with long openings through a process called facilitation. However, the mechanism whereby CaMK and the IQ domain are targeted to LTCCs is unknown. Endogenous CaMK is targeted to LTCCs in excised cell membrane patches because LTCC P(o) increased significantly in CaM-enriched (20 microM) bath solution and this effect was prevented by a specific CaMK inhibitory peptide, but not by an inactive control peptide.

Phosphodiesterase type 5 as a pharmacologic target in erectile dysfunction.

The scientific rationale of pharmacologically inhibiting phosphodiesterase type 5 (PDE5) in the treatment of erectile dysfunction (ED) is reviewed. Published literature on the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway for penile erection and on PDE5 inhibition using sildenafil as the model for pharmacologic PDE5 inhibition are assessed. The key second messenger in the mediation of penile erection is cGMP.

The caudal ganglionic eminence is a source of distinct cortical and subcortical cell populations.

During development, the mammalian ventral telencephalon is comprised of three major proliferative zones: the medial (MGE), lateral (LGE) and caudal (CGE) ganglionic eminences. Through gene expression studies, in vitro migration assays, genetic mutant analysis and in vivo fate mapping in mice, we found that the CGE is a progenitor region that is distinct from both the MGE and LGE. Notably, CGE cells showed a unique in vivo pattern of migration, and the CGE contributed cells to nuclei distinct from those populated by the MGE and LGE.

Dorsoventral patterning is established in the telencephalon of mutants lacking both Gli3 and Hedgehog signaling.

Considerable data suggest that sonic hedgehog (Shh) is both necessary and sufficient for the specification of ventral pattern throughout the nervous system, including the telencephalon. We show that the regional markers induced by Shh in the E9.0 telencephalon are dependent on the dorsoventral and anteroposterior position of ectopic Shh expression.

Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity.

Substrate binding to the phosphodiesterase-5 (PDE5) catalytic site increases cGMP binding to the regulatory domain (R domain). The latter promotes PDE5 phosphorylation by cyclic nucleotide-dependent protein kinases, which activates catalysis, enhances allosteric cGMP binding, and causes PDE5A1 to apparently elongate. A human PDE5A1 R domain fragment (Val(46)-Glu(539)) containing the phosphorylation site (Ser(102)) and allosteric cGMP-binding sites was studied.

Biological evidence that SOCS-2 can act either as an enhancer or suppressor of growth hormone signaling.

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH) signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling.

Pharmacology of phosphodiesterase-5 inhibitors.

The clinical properties (efficacy and safety profile) of a medicine are related not only to its mode of action, but also to its selectivity for its target (usually a receptor or enzyme) and are also influenced by its pharmacokinetic properties (absorption, distribution, metabolism and elimination). The growing number of phosphodiesterase inhibitors that are selective for phosphodiesterase-5 (PDE5) represent a promising new class of compounds that are useful for the treatment of erectile dysfunction and perhaps other disorders. Some of the basic pharmacodynamic and pharmacokinetic parameters that describe drug action are discussed with regard to the new PDE5 inhibitors.

Ectopic expression of Gcm1 induces congenital spinal cord abnormalities.

Brief ectopic expression of Gcm1 in mouse embryonic tail bud profoundly affects the development of the nervous system. All mice from 5 independently derived transgenic lines exhibited either one or both of two types of congenital spinal cord pathologies: failure of the neural tube to close (spina bifida) and multiple neural tubes (diastematomyelia). Because the transgene is expressed only in a restricted caudal region and only for a brief interval (E8.

A conserved serine juxtaposed to the pseudosubstrate site of type I cGMP-dependent protein kinase contributes strongly to autoinhibition and lower cGMP affinity.

Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type Ialpha and type Ibeta (PKG-Ialpha and PKG-Ibeta), respectively. Autophosphorylation of this residue is associated with activation of type I PKGs. To determine the role of this conserved serine, point mutations have been made in PKG-Ialpha (S64A, S64T, S64D, and S64N) and PKG-Ibeta (S79A).

Growth enhancement in suppressor of cytokine signaling 2 (SOCS-2)-deficient mice is dependent on signal transducer and activator of transcription 5b (STAT5b).

Mice lacking suppressor of cytokine signaling-2 (SOCS-2) exhibit accelerated postnatal growth resulting in adult mice that are 1.3 to 1.5 times the size of normal mice.

Mechanisms of autoinhibition in cyclic nucleotide-dependent protein kinases.

Cyclic AMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) are autoinhibited through multiple interactions between their respective regulatory and catalytic domains. A large portion of this autoinhibition occurs through interactions between residues within the catalytic domain and those within either a substrate-like sequence (-RRXSX-) or pseudosubstrate sequence (-RRXAX-) in the regulatory domains. These contacts effectively inhibit catalysis by blocking substrate binding.

Telencephalic cells take a tangent: non-radial migration in the mammalian forebrain.

During development of the mammalian telencephalon, cells migrate via diverse pathways to reach their final destinations. In the developing neocortex, projection neurons are generated from cells that migrate radially from the underlying ventricular zone. In contrast, subsets of cells that populate the ventral piriform cortex and olfactory bulb reach these sites by migrating long distances.