Convex grating types for concentric imaging spectrometers.

The properties of convex gratings fabricated by electron-beam lithography are investigated. Three grating types are shown. The first is a single-panel, true blazed grating in which the blaze angle stays constant relative to the local surface normal. This grating provides high peak efficiencies of approximately 88% in the first order and 85% in the second order. The second grating has two concentric panels, with each panel blazed at a different angle. This type permits flexibility in matching the grating response to a desired form. The third type has a groove shape that departs from the sawtooth blazed profile to increase the second-order bandwidth. All these types are difficult or impossible to produce with conventional techniques. The gratings compare favorably with conventional (holographic and ruled) types in terms of efficiency and scatter. Simple scalar models are shown to predict the wavelength response accurately. These gratings allow the optical designer to realize fully the considerable advantages of concentric spectrometer forms.

Steady-state vortex-screening solitons formed in biased photorefractive media.

We report the observation of steady-state photorefractive vortex-screening solitons. As a singly charged circular vortex nested on a broad beam propagates through a biased strontium barium niobate crystal, it self-traps in both transverse dimensions despite the inherent anisotropy of the photorefractive nonlinearity. When the vortex beam is a doughnut-shaped narrow beam, it breaks up into two elongated slices (with a self-defocusing nonlinearity) or into two focused filaments (with a self-focusing nonlinearity). We demonstrate the optical guidance of a probe beam in a circular waveguide induced by the self-trapped vortex.

Preincubation with creatine enhances levels of creatine phosphate and prevents anoxic damage in rat hippocampal slices.

We have investigated the relationship between energy metabolism, NMDA-receptor antagonism, and anoxic damage in vitro. Anoxic damage was assessed by measuring protein synthesis, defined as the incorporation of [14C]lysine into perchloric acid-insoluble tissue extracts. The concentrations of energy metabolites were measured by ion-exchange HPLC. Anoxia caused an inhibition of protein synthesis, a reduction in phosphocreatine and adenosine triphosphate, and extensive neuronal damage. The reduction of protein synthesis depended on the duration of anoxia and the time allowed for recovery. Preincubation with the creatine dose-dependently (0.03-3 mmol/L) increased baseline levels of phosphocreatine, reduced the anoxia-induced decline in phosphocreatine and adenosine triphosphate, prevented the impairment of protein synthesis, and reduced neuronal death. Incubation with (R,S)-3-guanidinobutyric acid, a synthetic analogue of creatine that cannot be phosphorylated, did not prevent the anoxia-induced impairment of protein synthesis and did not enhance the levels of phosphocreatine and adenosine triphosphate. Incubation with a combination of both creatine and the noncompetitive NMDA antagonist MK-801 provided complete protection. These results indicate that energy status is a major factor controlling anoxic damage in the rat hippocampal slice.

WAL 2014--a muscarinic agonist with preferential neuron-stimulating properties.

The ability of WAL 2014 to elicit muscarinic responses was investigated in various in vitro and in vivo models. In CHO cells transfected with human m1- or m3- receptor genes, WAL 2014 was clearly more effective in stimulating the M1-mediated PI response. In isolated tissue preparations, WAL 2014 exhibited full agonist properties in the rabbit vas deferens (putative M1 receptor) and behaved like a partial agonist at M2 receptors in the atrium and M3 receptors in the ileum of guinea-pigs. In the pithed rat, in which the increase in blood pressure is mediated through a stimulation of M1 receptors in sympathetic ganglia, WAL 2014 produced a full dose response curve, whereas the reference compounds RS 86 and arecoline exhibited a bell-shaped behaviour. This is in accord with the view that WAL 2014 selectively activates M1 receptors in sympathetic ganglia, whereas conventional agonists in the same dose range stimulate both ganglionic M1 and vascular M3 receptors. The preferential neuron-stimulating properties were confirmed by EEG recording in the rabbit, in which muscarinic activation occurred at doses similar to those for ganglionic stimulation in the pithed rat. On the other hand, higher doses of WAL 2014 were needed to elicit muscarinic effects in peripheral effector organs, i.e. bradycardia, urinary bladder contraction and increase in airway resistance. It is concluded that WAL 2014 due to its preferential neuronal activity is a promising candidate for a cholinergic substitution therapy in Alzheimer's disease.

Activation of excitatory amino acid receptors cannot alone account for anoxia-induced impairment of protein synthesis in rat hippocampal slices.

We have investigated the contribution of excitatory amino acid receptor activation to the inhibition of protein synthesis observed after anoxia in rat hippocampal slices. Protein synthesis was assessed in normoxic medium by measuring the incorporation of [14C]lysine into perchloric acid-insoluble tissue extracts. Protein synthesis was impaired after anoxia; the extent of inhibition was dependent on the duration of anoxia and on the time allowed for postanoxic recovery. There was a similar impairment under normoxic conditions when the N-methyl-D-aspartate (NMDA) receptor channel was activated by removing Mg2+ and adding NMDA. This was prevented by noncompetitive antagonists of the NMDA receptor channel (MK-801, phencyclidine, and N-allylnormetazocine). In contrast, incubation with the NMDA antagonists failed to prevent the protein synthesis inhibition caused by anoxia, although it moderately facilitated the postanoxic recovery. Protein synthesis was also impaired under normoxic conditions after incubation with quisqualate and kainate, agonists of non-NMDA glutamate receptors. This impairment was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of these receptors. Although 6-cyano-7-nitroquinoxaline-2,3-dione alone failed to prevent anoxic damage, when used in combination with an NMDA antagonist it did partially enhance the later recovery of protein synthesis. These results indicate that the activation of excitatory amino acid receptors cannot alone account for anoxia-induced impairment of protein synthesis in rat hippocampal slices.

Pramipexole, a dopamine D2 autoreceptor agonist, decreases the extracellular concentration of dopamine in vivo.

Pramipexole (SND 919) is a dopamine D2 autoreceptor agonist which is structurally related to talipexole (B-HT 920), a potential antipsychotic agent. The aim of this study was to investigate the effects of pramipexole on the extracellular concentration of dopamine in vivo. Dopamine and its metabolites, 3,4-dihydrophenylacetic acid and homovanillic acid, were measured in the anterior striatum of freely moving rats by microdialysis and high-performance liquid chromatography with electrochemical detection. Pramipexole (30 and 100 micrograms/kg) caused long-lasting decreases in the extracellular concentrations of dopamine and its metabolites. Talipexole (30 micrograms/kg) produced similar effects. Sulpiride (5 mg/kg), a selective dopamine D2 antagonist, caused a transient increase in the concentration of dopamine and long-lasting increases in the concentrations of its metabolites; it also reversed the effects of pramipexole. SCH-23390 (100 micrograms/kg), a selective dopamine D1 receptor antagonist, caused a transient increase in the concentration of dopamine but did not affect the concentrations of the metabolites. SCH-23390 failed to reverse the effects of pramipexole. These results indicate that pramipexole reduces the extracellular concentrations of dopamine and its metabolites in vivo through a reversible interaction with the dopamine D2 receptor.

Application and validation of an ion-exchange high-performance liquid chromatographic method for measuring adenine nucleotides, creatine and creatine phosphate in mouse brain.

We have modified and applied an ion-exchange high-performance liquid chromatographic method for measuring adenine nucleotides (adenosine monophosphate, adenosine diphosphate and adenosine triphosphate) as well as creatine and creatine phosphate in brain tissue. There was a linear relationship between the area of each peak and the amount of standard injected onto the column in the concentration range 0.5-25 nmol per 50 microliters. The concentrations of creatine phosphate and creatine were not stable in a standard mixture for 20 h at 4 degrees C unless the pH of the standard mixture was adjusted to neutral. We therefore strongly recommended the neutralization of all standard mixtures and samples before storage. The measurements of adenine nucleotides, creatine and phosphate in control mouse brain determined by this method agreed well with an enzymic method of nucleotide measurement. Furthermore, both methods detected similar decreases in the concentrations of adenosine triphosphate and creatine phosphate, together with concomitant increases in the concentrations of adenosine diphosphate, adenosine monophosphate and creatine when mice were placed under anoxic conditions (either 30 s or 2 min); these changes were greater after 2 min of anoxia than after 30 s of anoxia.

Central alpha-adrenoceptors during the development of hypertension in rats on high and low salt intake.

Our purpose was to investigate the binding characteristics of central alpha-adrenoceptors during the early stages of the development of hypertension in rats on high and low salt (NaCl) intake. We measured alpha 1-[( 3H]prazosin) and alpha 2-[( 3H]rauwolscine) binding in membranes of the hypothalamus and medulla oblongata of six groups of young Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR) and subtotally nephrectomized WKY (SN) rats with mean arterial blood pressure (MAP) ranging from normotensive to hypertensive levels after 1 week of salt restriction or loading. In the hypothalamus the SN-high salt rats and both SHR groups had elevated alpha 1-number but there was no change in alpha 2-number. Moreover, MAP was positively correlated with mean hypothalamic alpha 1-number in the six groups. In the medulla oblongata alpha 1-number was unaffected. However, high salt diet influenced medullary alpha 2-binding in the opposite manner in WKY rats versus SN rats and SHR. In these latter groups the affinity was increased and the number decreased in response to high salt intake. Furthermore, a positive correlation between MAP and mean alpha 1:alpha 2 ratio existed in both the hypothalamus and the medulla of the six groups. The data suggest that hypothalamic alpha 1-binding capacity was increased in SHR due principally to a genetic condition which is mimicked by salt loading in the SN rats. Medullary alpha 2-adrenoceptors of WKY, which remained normotensive despite salt loading, responded differently to high salt intake than those of the SN and SHR, whose blood pressure rose significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of 7 alpha, 17 alpha-dimethyl-19-nortestosterone (mibolerone) to androgen and progesterone receptors in human and animal tissues.

In rat uterus and prostate, 7 alpha, 17 alpha-dimethyl-19-nortestosterone (DMNT) binds to the androgen receptor specifically and with high affinity. However, this steroid does not bind to glucocorticoid receptors, since it does not displace binding of [3H]triamcinolone acetonide in calf thymus cytosol. In calf uterine and human breast tumor cytosols DMNT binds to the androgen and progesterone receptors, since binding of [3H] DMNT is displaced by unlabeled 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3, 20-dione triamcinolone acetonide, and 5 alpha-dihydrotestosterone (DHT). Conversely, binding of [3H]16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione is effectively competed for by unlabeled DMNT but not by DHT. The observed differences in binding of [3H]DMNT to rat and calf uterine cytosols suggest the species specificity of progesterone receptors. Unlike DHT, DMNT has no appreciable binding to human sex-steroid binding globulin. These findings suggest DMNT as a suitable ligand for measurement and characterization of androgen receptors in rat and human prostate.

Complete nucleotide sequences of Bacillus plasmids pUB110dB, pRBH1 and its copy mutants.

The deletion plasmids, pRBH1 (1.5 MDa, kanamycin resistance, Kmr) and pUB110dB (1.5 MDa, Kmr), were obtained from pTB913 (2.9 MDa, Kmr, isolated from a thermophilic bacillus) and pUB110 (3.0 MDa, Kmr, from Staphylococcus aureus), respectively. All the nucleotide sequences of these deletion plasmids were determined. Replication origin regions of pRBH1 and pUB110dB contained, respectively, 63 base-pair inverted repeat and a large open reading frame, encoding RepB protein (235 amino acid residues). The nucleotide sequences were identical to each other except for one base in the center of the inverted repeat. Two copy number mutant plasmids, pRBHC3 and pRBHC7, were obtained from pRBH1. The mutation points were located at different positions in the RepB protein coding region (Gly to Asp for pRBHC3 and Gly to Glu for pRBHC7). RepB protein was shown to be involved in the copy number control of these plasmids.

Changes in the sedimentation properties of cytosolic androgen receptors associated with activation in vitro and in vivo.

In cell-free systems androgen receptor (AR) labeled with (3H)DHT at 0 degrees C in the presence of 50mM molybdate remains unactivated (less than 3% binding to nuclei) and untransformed (7-8S on sucrose density gradients containing 0.4M KCl and 50mM molybdate). In the absence of molybdate, however, these complexes undergo activation and transformation even at 0 degrees C, albeit, very slowly. Incubation of unactivated, untransformed AR complexes at 18 degrees C, or at 0 degrees C in the presence of 0.4M KCl, greatly accelerated both activation and transformation. Activation and transformation are also associated with formation of high affinity (3H)DHT-receptor complexes as indicated by decreased rates of (3H)DHT dissociation from the receptor. Cytosolic AR complexes labeled with (3H)DHT in tissue slices at 37 degrees C, or in vivo, undergo rapid activation, transformation and nuclear translocation. The data suggest that activation and transformation of cytosolic AR in cell-free systems is associated with changes in the physicochemical properties of AR similar to those occurring upon hormone binding in intact cells and in vivo.

Interaction of estradiol and estriol with uterine estrogen receptor in vivo and in excised uteri or cell suspensions at 37 C: noncooperative estradiol binding and absence of estriol inhibition of estradiol-induced receptor activation and transformation.

The partial agonist and antagonist properties of estriol (E3) have been related to the brief nuclear retention of receptor-E3 complexes and to the lower affinity of E3 for the receptor compared to estradiol (E2). More recently, it was proposed that the partial agonist/antagonist activity of E3 may be due to its ability to eliminate positive cooperative binding of [3H]E2 to cytosolic estrogen receptor. In this model, positive cooperativity is related to receptor activation and transformation. We first examined the long term effects of E3 on E2 action in vivo. Mature ovariectomized rats were treated for 16 days with E2, E3, or mixtures of these two substances delivered through Alzet pumps at a constant hourly rate (E2, 0.04 microgram; E3, 0.4 and 0.04 microgram; E2 and E3, 0.04 and 0.4 microgram). The effects of E3 on uterine growth, induction of progesterone receptor synthesis, and activation (nuclear binding) of estrogen receptor suggest that when given continuously, E3 acts as a full agonist and does not inhibit E2 action. Furthermore, incubation of uteri at 37 C with [3H]E2 in the presence of a 1- to 20-fold molar excess of nonradioactive E3 did not alter the subcellular distribution of receptor-[3H]E2 complexes (80% nuclear and 20% cytosolic), demonstrating that E3 does not inhibit E2-induced receptor activation (i.e. the increased nuclear binding of receptor). Similarly, 4S to 5S transformation of [3H]E2-labeled estrogen receptor in intact uteri was not inhibited by E3. Equilibrium binding of [3H]E2 to uterine cell suspensions at physiological temperature (37 C) was noncooperative; nonradioactive E3 did not alter the affinity of the estrogen receptor for [3H]E2; Dixon plot analysis indicates that E3 is a purely competitive inhibitor of [3H]E2 binding. This, in conjunction with the lower affinity of the receptor for E3 than for E2, adequately explains the agonistic-antagonistic properties of E3.(ABSTRACT TRUNCATED AT 250 WORDS)

A new exchange procedure for the quantitation of prostatic androgen receptor complexes formed in vivo.

A procedure is described for the measurement of rat prostatic androgen receptor saturated in vivo with non-radioactive androgen. While NaSCN alone induces irreversible dissociation (denaturation) of androgen from the receptor, the combination of this chaotropic salt (0.15 M) with sucrose (15%) and sodium molybdate (10 mM) allows the exchange of R DHT with [3H]DHT at 0 degrees C with only minimal receptor denaturation. The validity of the present exchange assay is based on the following: a similar quantity of androgen receptor was detected when binding was measured directly after in vivo treatment with radioactive androgen or indirectly by [3H]DHT exchange after treatment with non-radioactive androgen. Steroid specificity, sedimentation analysis and equilibrium association constants indicated that this exchange assay labels the androgen receptor without interference from other prostatic steroid binding proteins. With this method it is now possible to quantitate not only prostatic androgen receptors bound to androgens in vitro but also hormone-receptor complexes formed in intact animals under the influence of endogenous androgen.

Resolution of non-activated and activated androgen receptors based on differences in their hydrodynamic properties.

This study shows that cytosolic androgen receptor of rat ventral prostate sediments at 10-11 S on conventional low salt sucrose density gradients (SDG), and at 4.6 S on high salt SDG, whether it is activated or not; inclusion of 10 mM Na2MoO4 in all buffers does not alter these sedimentation coefficients. In the presence of 50 mM Na2MoO4 non-activated and activated androgen receptors sediment in high salt SDG at 7-8 S and 4.6 S, respectively. Thus the presence of high concentrations of molybdate during centrifugation inhibits the KCl induced disaggregation of receptor into subunits. Similar effects are observed on Sephacryl-S200 gel filtration; in 50 mM MoO2-4 and 0.4 M KCl non-activated receptor has an estimated Stokes radius of 67 A; this value decreases to 52 A upon activation in the presence of proteolysis inhibitors; omission of molybdate during chromatography yielded 52 A and 27 A entities. Estimated mol. wts are 198,000 Daltons for the non-activated 67 A form and 98,000 Daltons for the activated 52 A receptor. Sodium molybdate (50 mM) prevents temperature (18 degrees C) and high ionic strength (0.4 M KCl) induced receptor activation. This inhibition was overcome by removing molybdate by centrifugal gel filtration, or by increasing the KCl concentration to 0.8 M. The inhibitory effects of molybdate on salt induced receptor disaggregation into activated subunits are no longer observed at pH greater than 7.4 or after chemical modification of sulfhydryl groups. Once androgen receptor has been disaggregated into its activated subunits the activated state is maintained even upon reassociation to 10-11 S aggregates in low salt. The relative concentrations of KCl and molybdate are critical; thus, 10 mM Na2MoO4/0.4 M KCl and 50 mM Na2MoO4/0.8-1.2 M KCl did not differentiate activated from non-activated androgen receptor based on their hydrodynamic properties. In the presence of 0.4 M KCl and 50 mM molybdate, however, the hydrodynamic properties of androgen receptor can be correlated with receptor activation.

Interaction of cyproterone acetate with rat prostatic androgen receptors.

We have investigated the binding of cyproterone acetate (CA) to cytosolic androgen receptors (RC) and translocation of the RCCA complex into the nucleus. In a cell-free system (3H)CA binds to cytosolic androgen receptors with high affinity (KD = 11.6 nM) and limited capacity (180-200 femtomoles/mg protein). (3H)CA, however, dissociates very rapidly from the cytosolic and nuclear androgen receptors (Rn) at 0 degree C. Incubation of RC (3H)CA at 20 degrees C increased its ability to bind to nuclei. Translocation of RC (3H)CA to nuclei of intact cells was demonstrated after incubation of prostatic tissue with (3H)CA in tissue culture medium at 37 degrees C. In vivo administration of CA to castrated rats promoted RCCA translocation but did not induce androgen receptor replenishment. These data demonstrate that CA binds to and translocates androgen receptors to nuclei without concomitant receptor replenishment.

Conversion of estrogen receptor from a state with low affinity for estradiol into a state of higher affinity does not require 4S to 5S dimerization.

This study was undertaken to establish whether the heat-promoted conversion of receptor-estradiol complex (RE2) from a state with fast into a state with slow E2 dissociation requires 8S/4S to 5S transformation. The calf uterine estrogen receptor labeled with [3H]E2 at 0 C (state with low affinity for E2) was immobilized on hydroxylapatite (HAP) in the absence (8S oligomer) or presence (4S monomer) of 0.4 M KCl and heated at 28 C in the presence of unlabeled diethylstilbestrol. Under these conditions, the dissociation of [3H]E2 was biphasic and occurred at rates similar to those obtained with R[3H]E2 free in cytosol. In contrast to the latter, however, the heat-promoted conversion of HAP-immobilized R[3H]E2 into a state of higher affinity for E2 was not accompanied by receptor dimerization, since the HAP eluate (0.4 M phosphate buffer) contained only the 4S monomer; upon reheating or desalting of this eluate, 4S to 5S dimerization occurred. The heat-promoted formation of 4S RE2 monomers with higher affinity for E2 was not due to monomer-HAP interactions, since even after elution from HAP, the 4S RE2 remained in the state of higher affinity for E2. Addition of pyridoxal 5'-phosphate to slow dissociating, high affinity 5S R[3H]E2 dimers free in cytosol induced rapid [3H]E2 dissociation, although the receptor remained unaltered in the transformed dimerized state. The effect of PLP was readily reversed by the addition of lysine. It is proposed that the 4S receptor monomers exist in two conformational states; upon E2 binding to the low affinity state, conformational changes result in stronger interactions between the steroid and the amino acid residues of the estrogen-binding domain; thus, the rate of E2 dissociation decreases. The formation of this 4S RE2 state with higher affinity for E2 is independent from receptor dimerization. A model is presented in which 4S to 5S transformation may lead to stabilization of 4S monomers in the conformation with higher affinity for E2.

The synthesis of ribosomal RNA and ribosomal protein and their incorporation into ribosomes in the uterus of the oestrogen-stimulated immature rat.

The effect of oestrogen on the synthesis of ribosomal proteins in the uterus of the immature rat has been investigated. Stimulated synthesis peaks, at 6-7-times control levels, 12 h after a single administration of the hormone. The stimulated synthesis and incorporation of newly made proteins into ribosomal particles exhibit very similar kinetics. The incorporation of newly made rRNA into ribosomes mirrors that of ribosomal protein but lags several hours behind the peak of oestrogen-stimulated rRNA synthesis.

The effects of oestradiol-17 beta on the synthesis and modification of ribosomal proteins in the uterus of the immature rat.

The effects of oestrogen on the incorporation of newly-made ribosomal proteins into the ribosomes of the immature rat uterus has been investigated. Different newly-made proteins were shown to enter ribosomes at different rates and there was some evidence that the hormone exerted differential effects. Oestradiol also stimulated the phosphorylation of ribosomal protein S6 but the effect could be explained by hormone-induced changes in the precursor pools.

Differences in the physicochemical characteristics of androgen-receptor complexes formed in vivo and in vitro.

This study compares the physicochemical characteristics of the androgen-receptor hormone complexes formed in vitro by incubation of prostatic cytosol with tritiated 5 alpha-dihydrotestosterone (DHT) and methyltrienolone (R1881; 17 beta-hydroxy-17 alpha-methyl-4,9, 11-estra-trien-3-one) with those of hormone-receptor complexes formed in vivo upon hormone injection. [3H]DHT and [3H]R1881 had similar affinities for the androgen receptor in vitro (Kd = 0.3 nM). Dissociation of DHT at 0 C from the receptor complexes formed in vitro or in vivo was much slower than that of R1881. Furthermore, DHT and R1881 dissociated much more slowly from the cytoplasmic receptor labeled in vivo than in vitro. The sedimentation characteristics of the in vitro and in vivo formed hormone-receptor complexes were similar when analyzed on sucrose density gradients containing 400 mM KCl and 10 mM Na2MoO4. Higher concentrations (50 mM) of Na2MoO4, however, prevented the salt-induced disaggregation of the in vitro formed receptor complexes, which sedimented at 7-8S. In contrast, androgen-receptor complexes formed in vivo sedimented as 5.5S complexes, even in the presence of 50 mM molybdate. These differences were paralleled by the elution patterns from Sephacryl S-200. A further difference was found in the sensitivity of the hormone-receptor complex to the organic mercurial reagent mersalyl acid. This reagent, at 0.2 mM, induced ligand exchange of 80-90% of the in vitro formed hormone-receptor complexes, whereas it was nearly ineffective with complexes formed in vivo. Finally, the prostatic androgen receptor content 1 h after injection of radioactive steroid into castrated rats was 12-14 pmol/mg DNA, while incubation of tissue slices at 37 C yielded only 3-4 pmol receptor/mg DNA.