Unlocking Lewis acidity via the redox non-innocence of a phenothiazine-substituted borane.

We describe a new approach to enhancing Lewis acidity, through the single electron oxidation of a borane with a pendant phenothiazine. This results in the formation of a persistent radical cation with increased electrophilicity. Computational and experimental studies indicate this radical cation significantly enhances the Lewis acidity and catalytic activity compared to its neutral analog. These results illustrate the viability of this approach in turning on the Lewis acidity of relatively inert boranes.

Selective dimerization of α-methylstyrene by tunable bis(catecholato)germane Lewis acid catalysts.

The synthesis of a variety of bis(catecholato)germanes is reported. The Lewis acidity of the bis(catecholato)germanes was assessed using the experimental Gutmann-Beckett method and computational FIA and GEI methods. The oligomerization of alkenes using bis(catecholato)germanes demonstrates the use of these complexes in catalysis. The use of donor additives in the dimerization of α-methylstyrene resulted in selectivity control comparable to transition metal catalyst systems.

Light and propranolol suppress the nocturnal elevation of serotonin in the cerebrospinal fluid of rhesus monkeys.

Markedly elevated nighttime concentrations of serotonin in rhesus monkey cerebrospinal fluid were reduced to daytime levels by exposing the monkeys to continuous light or to the beta-adrenergic antagonist propranolol. Nighttime elevations of melatonin in cerebrospinal fluid were also suppressed by propranolol and light. Serotonin released in large quantities at night appears to be regulated like melatonin, and may act as a cerebroventricular hormone to influence brain and pituitary function at night.

Simulated anchoring of a nematic liquid crystal at a polymer surface.

Liquid-crystal anchoring at a polymer surface arises from interactions at several different length scales. At the molecular level, a liquid-crystal molecule may tend to align with the substrate polymer chain, while at the nanometer length scale grooves can exist that arise from the periodic repeat structure of a polymer chain or from nanometer-scale undulations due to surface stresses. On a still longer scale there is the secondary effect of grooves or surface inhomogeneities. We have performed a total of more than 900 ns of atomistic molecular dynamics simulations in order to study the relative importance of the molecular-level interaction and the topography of the polymer surface in liquid-crystal anchoring. Substrates were constructed in which grooves were induced along a direction perpendicular to the constituent molecular chains. In the results presented for the case of 32 5CB molecules on a poly(vinyl alcohol) substrate, the liquid-crystal director orientation appeared to be determined principally by the substrate chain orientation. Only for the deepest grooves did the director align along the grooves and perpendicular to the substrate molecular chain direction.

Range of interlayer interactions in smectic- C liquid crystals.

The fact that the elastic constant for bending a layer of smectic- C liquid crystal along its c director differs from the value for bending in the perpendicular direction has recently been shown to give rise to interactions between distant layers. The effect of this entropy-induced interaction is to favor a parallel or antiparallel alignment of the c directors in these nonadjacent layers. We calculate in detail the range and strength of this interaction in both infinite and finite samples, and find the results to depend mainly on the ratio of the average layer bending elastic constant to the layer compression modulus. At low values of this ratio, the interlayer interaction is of long range in a bulk sample, while at high values of the ratio it decays as the inverse cube of the interlayer distance. For a sample confined between rigid substrates parallel to the layers, the interaction is greatly reduced. For a free-standing film the interaction may be enhanced if the surface tension is weak, but may be diminished if the surface tension is strong.

Impact of heat on nanocrystalline silver dressings. Part I: Chemical and biological properties.

Thermal stability of heat-treated nanocrystalline silver dressings was investigated using chemical techniques and biological assays. Dressings were heat-treated for 24h at temperatures from 23 to 110 degrees C. Bactericidal efficacy of heat-treated dressings was measured using a log reduction assay, while antibacterial longevity was determined via plate-to-plate transfer corrected zone of inhibition assays. Over the temperature range tested, biological activity dropped from excellent to negligible. Biological longevity results showed that controlled release properties of the dressings were significantly reduced by heat treatments above 75 degrees C. These data illustrate nanocrystalline silver sensitivity to heat. Further, it was clear that dressing efficacy is determined by total available soluble silver, not total silver in the dressing. It was determined that the quantity of soluble silver decreased significantly with increased heat treatment temperatures. These results should be considered in developing new nanocrystalline drug delivery systems.

Impact of heat on nanocrystalline silver dressings. Part II: Physical properties.

This work explores the effects of elevated temperature on the physical and chemical properties of nanocrystalline silver, and relates it to previously observed thermally induced changes in biological activity [Taylor PL et al. Biomaterials, in press, doi:10.1016/j.biomaterials.2005.05.040]. Microstructural evolution of nanocrystalline silver dressings, heat-treated for 24 h at temperatures from 23 to 110 degrees C, was studied in detail using X-ray diffraction (XRD), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). These analyses indicated that silver nanocrystalline coatings undergo significant changes in structure when exposed to elevated temperature. XRD analysis showed a rapid increase in crystallite size above 75 degrees C along with decomposition of crystalline silver oxide (Ag2O) at the onset of crystallite growth. SEM imaging showed a loss of fine features and sintering of the structure at elevated temperatures. The XPS data indicated that silver-oxygen bonds disappeared completely, with the initial decomposition occurring between 23 and 37 degrees C, and total oxygen in the coating decreased from 16-17% to 6.5% over the temperature range of 75-110 degrees C. A comparison of these results to the data of Taylor et al. [Biomaterials, in press, doi:10.1016/j.biomaterials.2005.05.040] indicates that the unique biological properties of nanocrystalline silver are related to its nanostructure. This should guide future development of therapeutic nanocrystalline silver delivery systems.

Chloride channels mediate the response to gonadotropin-releasing hormone (GnRH) in Xenopus oocytes injected with rat anterior pituitary mRNA.

Functional expression of receptors for GnRH was studied using Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat anterior pituitary glands. Whole-cell currents were monitored using two-electrode voltage-clamp techniques. In oocytes which responded to both GnRH and TRH, the GnRH response showed a longer latency and time-to-peak than the TRH response. The response to GnRH or an agonist of GnRH receptors, buserelin (1 nM-1 microM) consisted of current fluctuations and occurred in a dose-dependent manner. This GnRH response was blocked by the Cl- channel blockers 9-AC (9-anthracene carboxylic acid; 1 mM), 4,4'-diisothiocyanastilbene-2,2'-disulfonic acid (0.1 mM), and diphenylamine-2-carboxylic acid (0.1 mM). The reversal potential for the GnRH-induced current fluctuations was -25 mV, comparable with the reported Cl- equilibrium potential in Xenopus oocytes, and its shift, when the external concentration of Cl- was changed, was reasonably described by the Nernst equation. These results indicate that the GnRH-induced response was dependent on the activity of Cl- channels. Ca2+ also plays a role, as the GnRH-induced response was reversibly suppressed by a calmodulin inhibitor, chlordiazepoxide (0.2 microM), and by a blocker of intracellular Ca2+ release, TMB-8 (8-(N.N-diethylamino) octyl-3,4,5-trimethoxybenzoate; 0.1-0.2 mM). It is concluded that GnRH (and TRH) receptors, expressed in Xenopus oocytes by injecting exogenous mRNA from rat anterior pituitary glands, operate via activation of Ca2+-dependent Cl- channels.

Agonist-induced endocytosis and recycling of the gonadotropin-releasing hormone receptor: effect of beta-arrestin on internalization kinetics.

This study examined the dynamics of endocytotic and recycling events associated with the GnRH receptor, a unique G protein-coupled receptor (GPCR) without the intracellular carboxyl-terminal tail, after agonist stimulation, and investigated the role of beta-arrestin in this process. Subcellular location of fluorescently labeled epitope-tagged GnRH receptors stably expressed in HEK 293 cells was monitored by confocal microscopy, and the receptor/ligand internalization process was quantified using radioligand binding and ELISA. Agonist stimulation resulted in reversible receptor redistribution from the plasma membrane into the cytoplasmic compartment, and colocalization of internalized GnRH receptors with transferrin receptors was observed. Internalization experiments for the GnRH receptor and another GPCR possessing a carboxy-terminal tail, the TRH receptor, showed that the rate of internalization for the GnRH receptor was much slower than for the TRH receptor when expressed in both HEK 293 and COS-7 cells. TRH receptor internalization could be substantially increased by coexpression with beta-arrestin in COS-7 cells, while GnRH receptor internalization was not affected by coexpression with beta-arrestin in either cell type. Coexpression of the GnRH receptor with the dominant negative beta-arrestin (319-418) mutant did not affect its ability to internalize, and activated GnRH receptors did not induce time-dependent redistribution of beta-arrestin/green fluorescent protein to the plasma membrane. However, the beta-arrestin mutant impaired the internalization of the TRH receptor, and activated TRH receptors induced the beta-arrestin/green fluorescent protein translocation. This study demonstrates that, despite having no intracellular carboxy-terminal tail, the GnRH receptor undergoes agonist-stimulated internalization displaying distinctive characteristics described for other GPCRs that internalize via a clathrin-dependent mechanism and recycle through an acidified endosomal compartment. However, our data indicate that the GnRH receptor may utilize a beta-arrestin-independent endocytotic pathway.

Phase sequences and long-range interactions in ferroelectric liquid crystals.

The origin of the long-range interlayer interactions responsible for the variety of phases exhibited by ferroelectric liquid crystals is discussed. It is shown that the anisotropy of the elastic constants that govern layer bending in smectic- C liquid crystals results in an effective long-range interaction between the smectic layers. The nature of this interaction is such as to favor a mutual alignment of the c directors of the layers in either a parallel or antiparallel orientation. The free energy of the system is the sum of the contributions of these long-range interlayer interactions and the short-range interaction between nearest-neighboring layers, which favors a purely helical structure for the c directors. The long-range interaction is found to favor commensurate structures while the short-range term favors incommensurate helices. The resulting structure is of the type characterized in the "distorted clock model." The phase diagrams that result from the application of this theory are consistent with the experimentally observed phase sequences.

The rat gonadotropin-releasing hormone receptor internalizes via a beta-arrestin-independent, but dynamin-dependent, pathway: addition of a carboxyl-terminal tail confers beta-arrestin dependency.

This study examined the mechanism underlying the rat GnRH receptor (GnRH-R) internalization pathway by investigating the role of added/extended C-terminal tails and the effect of beta-arrestins and dynamin. The internalization of the wild-type (WT) rat GnRH-R, stop codon mutants, GnRH-R/TRH receptor (TRH-R) chimera, rat TRH-R, and catfish GnRH-R was examined using radioligand binding assay. Overexpression of beta-arrestin in COS-7 cells expressing each of the receptor constructs substantially increased endocytosis rate constants (k(e)) of the TRH-R, catfish GnRH-R, and GnRH-R/TRH-R chimera, but not of the WT rat GnRH-R and stop codon mutants. The beta-arrestin-promoted increase in the k(e) value was diminished by cotransfecting cells with the dominant negative beta-arrestin-(319-418) mutant, whereas WT GnRH-R and stop codon mutant internalization were unaffected. Additionally, confocal microscopy showed that activated GnRH-Rs failed to induce time-dependent redistribution of either beta-arrestin-1- or beta-arrestin-2-green fluorescent protein conjugate to the plasma membrane. However, the dominant negative dynamin (DynK44A) mutant impaired internalization of all of the receptors regardless of their beta-arrestin dependency, indicating that they internalize via a clathrin-mediated pathway. We conclude that the mammalian GnRH-R uses a beta-arrestin-independent, dynamin-dependent internalization mechanism distinct from that employed by the other receptors studied.

Cloning, sequencing and tissue distribution of a candidate G protein-coupled receptor from rat pituitary gland.

A new member of the family of G protein-coupled receptors has been isolated from a rat pituitary cDNA library by the polymerase chain reaction (PCR) using degenerate oligonucleotide primers. The corresponding protein sequence shows seven transmembrane domains and contains conserved regions of homology characteristic of the G protein-coupled class of receptors. The novel receptor mRNA is expressed in the brain, pituitary gland and testis, and has been localized by in situ hybridization in discrete regions of the brain. Expression of the receptor mRNA in Xenopus oocytes and in transfected mammalian cells has not yet permitted identification of the corresponding ligand for this receptor.

Characterization of the mouse thyrotrophin-releasing hormone receptor gene: an exon corresponds to a deletion in the rat cDNA.

The cloning and characterization of the mouse TRH receptor (TRH-R) gene revealed an untranslated exon (exon 1), a single intron and an upstream dinucleotide repeat sequence (d(TG)16.d(AG)21) in the 5' untranslated region (UTR). The coding region was contained almost entirely on a second exon (exon 2), with the final amino acid and stop codon at the COOH terminus of the gene encoded by a third exon (exon 3) flanked by two introns. The 3' UTR was contained on the remainder of exon 3 and on the final exon (exon 4). Exon 3 (228 bp) corresponds exactly to a 228 bp deletion that exists in the rat TRH-R cDNA, but not in the mouse cDNA. The mouse TRH-R cDNA encodes a protein of 393 amino acids which is 96% homologous to the rat TRH-R protein of 412 amino acids, but is 19 amino acids shorter at its COOH terminus. The coding sequence for these 19 amino acids (plus 1 extra amino acid) does exist in the mouse TRH-R gene, but the sequence is encoded by exon 4, separated from the rest of the coding region by the stop codon and 223 bp of 3' UTR on exon 3. Splicing of exon 3 in the mouse TRH-R gene would remove the last amino acid, the stop codon and the 223 bp of 3' UTR, allowing transcription to continue into the 3' UTR on exon 4, which encodes the 19 extra amino acids found in the rat cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland.

We have isolated the TRH receptor (TRH-R) from a rat anterior pituitary cDNA library, determined its sequence and examined its functional characteristics. Expression studies were carried out in Xenopus oocytes and in COS-7 cells. Microinjection of sense RNA transcripts into Xenopus oocytes showed electrophysiological responses of between 800 and 1000 nA under voltage-clamp conditions. COS-7 cells were transiently transfected with the cDNA clone under the control of a cytomegalovirus promoter and inositol phosphate (IP) measurements carried out. In TRH-R transfected cells, TRH (100 nM) produced an approximately twofold increase in total IP production. In-situ hybridization in the rat anterior pituitary revealed a heterogeneous distribution of label, a characteristic pattern of TRH-R expression. The rat 3.3 kb insert coded for a protein of 411 amino acids compared with 393 for the mouse protein. Over its length, the rat TRH-R protein showed considerable homology with that of the mouse, except for a deletion of 232 bp in the 3'-coding region. This deletion did not appear to affect the functional characteristics of the receptor, as shown by electrophysiological studies with Xenopus oocytes and by transfection of the cDNA into COS-7 cells. The sequence given for the 3'-untranslated region is 1.5 kb longer than that reported for the mouse receptor, and extends to the poly(A) tail.

Functional expression of rat pituitary gonadotrophin-releasing hormone receptors in Xenopus oocytes.

Expression of receptors for the hypothalamic regulatory peptide, gonadotrophin-releasing hormone (GnRH), was investigated by intracellular recording from Xenopus oocytes injected with poly(A)+ mRNA isolated from rat anterior pituitary glands. Membrane depolarizations were induced in oocytes in a dose-dependent fashion following the application of GnRH (10nM - 1 microM) or a GnRH superactive agonist, buserelin (1nM - 1 microM). The response was reversibly blocked by the addition of a GnRH antagonist (1 microM). TRH (10nM - 1 microM) had no effect on most of these oocytes. In contrast, some other oocytes which showed no responses to GnRH or to the GnRH agonist, displayed depolarizing responses to TRH (10nM - 1 microM). A relatively small number of oocytes responded to both ligands. Control oocytes did not respond to the GnRH analogues or to TRH. This successful expression of the GnRH receptor could provide a new approach to the study of the receptor, and serve as a means for the isolation and cloning of the encoding genes.

Effect of small doses of bovine follicular fluid on the tonic secretion of gonadotrophins in the ewe.

Previous work has shown that treatment of ewes with steroid-free bovine follicular fluid (bFF), a rich source of inhibin, partially inhibits the increase in mean plasma concentrations of LH induced by ovariectomy. The present experiment was designed to test the hypothesis that this effect was a reflection of reduced LH pulse amplitude which would only be expressed at high (pharmacological) doses of bFF. To do this, we assessed the dose-response to bFF of the secretion of FSH and LH pulses in intact and acutely ovariectomized ewes. In intact ewes, a low dose of bFF (0.2 ml s.c. every 8 h) had no detectable effect on the secretion of FSH, an intermediate dose (0.6 ml s.c. every 8 h) depressed FSH concentrations for about 24 h and a high dose (1.8 ml s.c. every 8 h) reduced FSH concentrations to undetectable levels. In ewes treated with 1.8 ml bFF, FSH concentrations also remained undetectable after ovariectomy and did not increase until treatment was withdrawn. In ewes treated with 0.6 ml bFF, FSH concentrations were maintained at normal intact levels for about 32 h following ovariectomy but then rose to normal ovariectomized levels. In ewes treated with 0.2 ml bFF, FSH concentrations increased immediately after ovariectomy but more slowly than in control ovariectomized ewes. Profiles of LH pulses were recorded after ovariectomy, during and after the withdrawal of bFF treatment. In ewes treated with the highest dose (1.8 ml s.c. every 8 h), mean LH levels and pulse amplitude were lower than in control ewes and increased significantly following withdrawal of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

LHRH pulse frequency in normal and infertile men.

The aim of this study was to examine the hypothesis that decreased LHRH pulse frequency may be responsible for the preferential rise in FSH in infertile men. The LH pulse pattern was determined as an index of hypothalamic LHRH secretion in 21 infertile patients with idiopathic azoospermia or oligoasthenozoospermia and 14 fertile age-matched controls by frequent blood sampling at 10-min intervals for 24 h. The infertile patients were further divided into three groups according to their relative concentrations of FSH and LH: (1) normal FSH and LH, (2) raised FSH but normal LH, and (3) raised FSH and LH. LH pulses were detected by a computerized algorithm (Munro) validated against a threshold method. Concentrations of FSH, testosterone, sex hormone-binding globulin and oestradiol were measured in pooled plasma. Luteinizing hormone pulse frequencies in normal men were not significantly different from the infertile group as a whole. Similarly, mean LH pulse frequencies in infertile subgroups 1, 2 and 3 were not significantly lower than normal. Pulse interval, however, was increased in subgroup 1 compared with normal. Mean 24 h LH in group 2 was significantly higher than normal, but still within the normal range. The total testosterone, but not the free testosterone index was significantly decreased in the infertile group compared with normal. There was no correlation between mean FSH and LH pulse frequency or interval. In conclusion, our results show that in patients with seminiferous tubular dysfunction, the typical pattern of raised plasma FSH, increased LH pulse amplitude, raised FSH: LH ratio and normal or marginally low testosterone was not associated with any significant deviations in LHRH pulse frequency from the range observed in normal fertile men. This is not compatible with the hypothesis that decreased LHRH pulse frequency is associated with or the cause of the preferential rise in FSH in men with idiopathic infertility. Thus unlike anovulatory infertility in females, functional defects of hypothalamic LHRH secretion remain an uncommon finding in male infertility. Attempts to treat idiopathic oligozoospermia by altering LHRH pulse frequency is therefore unlikely to yield any clinical benefit.

Effects of Asn87 and Asp318 mutations on ligand binding and signal transduction in the rat GnRH receptor.

The gonadotrophin-releasing hormone (GnRH) receptor is unlike other G-protein coupled receptors in that the highly conserved amino acids, Asp in the second transmembrane region and Asn in the seventh, are interchanged. Site-directed mutagenesis studies mutated these residues back to their normally conserved positions. Two single mutants Asn87Asp & Asp318Asn and one double mutant Asn87Asp Asp318Asn were transiently expressed in COS-1 cells and their effect on binding to GnRH and inositol phosphate production measured. The single mutant Asp318Asn had no effect on ligand binding but abolished GnRH-dependent inositol phosphate production, whereas mutations Asn87Asp and Asn87Asp Asp318Asn show a complete loss of GnRH binding and subsequent inactivation of its second messenger system.

The roles of inhibin and gonadotrophin-releasing hormone in the control of gonadotrophin secretion in the ewe.

The respective roles and relative importance of ovarian inhibition and hypothalamic stimulation in the differential control of the secretion of FSH and LH were studied in the ewe. In the first experiment two groups of ten intact ewes were injected i.v. twice daily with 9 ml charcoal-extracted bovine follicular fluid (bFF), a preparation rich in inhibin (3.65 ku./ml), throughout the luteal phase of the oestrous cycle. Compared with the control ewes, this treatment significantly reduced pituitary and plasma FSH concentrations and increased the frequency and amplitude of the LH pulses, but did not affect pituitary LH concentrations. In a second experiment, five control and five bFF-treated ewes from experiment 1 were ovariectomized and the injection regime was altered to 2.5 ml s.c. every 8 h. This treatment was maintained for 21 days. In control ewes, plasma FSH concentrations rose significantly within 12 h and continued to rise for 3-4 days. Treatment with bFF abolished this increase and maintained plasma FSH concentrations below those observed in intact ewes. The rise in mean plasma LH concentrations evoked by ovariectomy was also partially inhibited in the bFF-treated ewes. The response to the gonadotrophin-releasing hormone (GnRH) agonist buserelin (5 micrograms i.v.) was measured 6, 12 and 18 days after ovariectomy. In control ewes the agonist consistently evoked large surges of both hormones but in bFF-treated ewes the FSH response was completely blocked and the initial phase of the LH response (the first 'pool') was greatly reduced. In experiment 3, six ewes were ovariectomized and passively immunized against GnRH 3 days after oestrus. The increase in plasma LH which normally follows ovariectomy was completely abolished and mean concentrations remained very low and did not change over the following 14 days. In contrast, mean FSH concentrations rose significantly within 12 h of ovariectomy and continued to rise until the third day, after which they fell gradually. Treating three of the ewes with bFF (2.5 ml s.c. every 8 h) 8 days after ovariectomy and immunization further reduced the FSH concentrations. When the ewes were injected repeatedly (200 ng i.v., hourly for 5 h) with [D-penicillamine-(But)6]-GnRH(1-9)nonapeptide-ethylamide, a synthetic GnRH analogue which does not bind to the antiserum, there was a rapid rise in the secretion of LH in both control and bFF-treated animals but, as with the responses to buserelin, the initial response was significantly lower in bFF-treated than in control ewes.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and characterisation of the marmoset gonadotrophin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe.

In order to facilitate the understanding of gonadotrophin-releasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly(10), [d-Trp(6)]-GnRH binding, with a K(d) value similar to those of both the rat and human forms, but with a greatly reduced B(max) value. The ED(50) for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but the maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser(140)Phe rat GnRH-R mutant was generated. The mutant had a K(d) value similar to that of the wild-type rat receptor, although the B(max) value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED(50) value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser(140)Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events.