High-affinity σ1 protein agonist reduces clinical and pathological signs of experimental autoimmune encephalomyelitis.

BACKGROUND AND PURPOSE: Selective agonists of the sigma-1 receptor (σ1 protein) are generally reported to protect against neuronal damage and modulate oligodendrocyte differentiation. Human and rodent lymphocytes possess saturable, high-affinity binding sites for compounds binding to the σ1 protein and potential immunomodulatory properties have been described for σ1 protein ligands. Experimental autoimmune encephalomyelitis (EAE) is recognized as a valuable model of the inflammatory aspects of multiple sclerosis (MS). Here, we have assessed the role of a σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, in EAE. EXPERIMENTAL APPROACH: EAE was induced in SJL/J female mice by active immunization with myelin proteolipid protein (PLP)139-151 peptide. The σ1 protein agonist was injected i.p. at the time of immunization (day 0). Disease severity was assessed clinically and by histopathological evaluation of the CNS. Phenotyping of B-cell subsets and regulatory T-cells were performed by flow cytometry in spleen and cervical lymph nodes. KEY RESULTS: Prophylactic treatment of EAE mice with the σ1 protein agonist prevented mononuclear cell accumulation and demyelination in brain and spinal cord and increased T2 B-cells and regulatory T-cells, resulting in an overall reduction in the clinical progression of EAE. CONCLUSIONS AND IMPLICATIONS: This σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, decreased the magnitude of inflammation in EAE. This effect was associated with increased proportions of B-cell subsets and regulatory T-cells with potential immunoregulatory functions. Targeting of the σ1 protein might thus provide new therapeutic opportunities in MS.

New synthesis of tic-hydantoins sigma-1 ligands and pharmacological evaluation on cocaine-induced stimulant effects.

Activation of the newly identified σ1 chaperone protein is involved in several aspects of the psychostimulant and addictive effects of cocaine. The development of ligands that selectively target the σ1 protein may lead to putative potent anti-cocaine agents. We report here a new and more convergent synthetic pathway to amino side chain substituted hydantoins. Twenty new analogs of our lead compound were synthesized. None of them showed better in vitro affinity for σ1 receptor than our lead compound. Nevertheless, three of them, obtained as racemates, showed high in vitro affinity and selectivity for σ1 receptor. A preliminary in vivo evaluation of their pharmacological activity identified compound 22 as one that increases cocaine-induced locomotor stimulation and therefore acts as a potential efficient σ1 agonist.

Separation of Tic-hydantoin enantiomers, potent sigma-1 agonists, by high performance liquid chromatography and capillary electrophoresis.

Stereospecific separations of seven Tic-hydantoin sigma-1 agonists were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and capillary electrophoresis (CE) method using neutral and anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (R(s)>3.3 with analysis times<25min) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. CE was used as an alternative technique to HPLC for the Tic-hydantoin derivatives separation. The enantiomers were fully resolved with highly sulfated beta-cyclodextrins at pH 2.5 (R(s)>1.5 with analysis times <11min). Both methods were validated in terms of linearity, detection and quantification limits. They were used to check the enantiomeric purity of the enantiomers.

Formulation and characterization of polyphenol-loaded lipid nanocapsules.

The purpose of this study was to design and characterize two flavonoid-loaded lipid nanocapsules (LNC) by applying the phase inversion process, and to enhance their apparent solubility and/or the stability. The flavonoid-loaded LNC were characterized by particle size, encapsulation efficiency, drug leakage rates, stability and spectroscopic studies. It was observed that quercetin-loaded LNC30 (3%) and LNC60 (2%) carried a particle size of 30.3 and 55.1 nm, respectively and significant higher entrapment efficiency. Encapsulation of quercetin (QC) in LNC enabled us to increase its apparent aqueous solubility by a factor of 100. And in view of calculations and results, it seems most probable that QC is arranged at this LNC interface between the oil phase and the hydrophilic polyethylene glycol moieties of the surfactant. In addition, colloidal suspensions proved to be stable in term of encapsulation for at least 10 weeks and QC was not oxidised. With simple chemical modification of (-)-epigallocatechin-3-gallate or (-)-EGCG, it was possible to reach very high encapsulation rates (95%). Thus we obtained stable colloidal suspensions of (-)-EGCG in water over 4 weeks while free (-)-EGCG solubilised in water exhibited 100% degradation within 4h. The initial problems (solubility and stability) of these flavonoids were resolved thanks to drug-loaded LNC.

Synthesis and antimalarial activity of new amino analogues of amodiaquine.

Amodiaquine remains one of the most prescribed antimalarial 4-aminoquinoline. To assess the importance of the 4'-hydroxyl group and subsequent hydrogen bond in the antimalarial activity of amodiaquine (AQ), a series of new analogues in which this functionality was replaced by various amino groups was synthesized. The incorporation of a 3'-pyrrolidinamino group instead of the 3'-diethylamino function of AQ allowed the development of a parallel series of amopyroquine derivatives. The compounds were screened against both chloroquine (CQ)-sensitive and -resistant strains of Plasmodium falciparum and their cytotoxicity evaluated upon the MRC5 cell line. Antimalarial activity in a low nanomolar range was recorded showing that the 4'-hydroxy function can be successfully replaced by various amino substituents in terms of activity without any influence of the level of CQ-resistance of the strains. Furthermore the ability of the compounds to inhibit beta-hematin formation was measured in order to discuss the mechanism of action of these new compounds. Compounds 7d and 8d exhibit a high selectivity index and may be considered as promising leads for further development.

Using Support Vector Machine Regression to Model the Retention of Peptides in Immobilized Metal-affinity Chromatography.

Retention of histidine-containing peptides in immobilized metal-affinity chromatography (IMAC) has been studied using several hundred model peptides. Retention in a Nickel column is primarily driven by the number of histidine residues; however, the amino acid composition of the peptide also plays a significant role. A regression model based on support vector machines was used to learn and subsequently predict the relationship between the amino acid composition and the retention time on a Nickel column. The model was predominantly governed by the count of the histidine residues, and the isoelectric point of the peptide.

Design, synthesis and antimalarial activity of a glyoxylylhydrazone library.

Synthesis of a new family of quinolylhydrazone derivatives and evaluation of their activity against a chloroquine-resistant strain of Plasmodium falciparum are described. The best compound displayed an activity 6-fold higher than chloroquine. None of the active compounds were found to inhibit beta-hematin formation in vitro in the same range as chloroquine and five among them displayed lower calculated vacuolar accumulation ratios, suggesting the implication of a different mechanism of action.

T-tubule localization of the inward-rectifier K(+) channel in mouse ventricular myocytes: a role in K(+) accumulation.

1. The properties of the slow inward 'tail currents' (I(tail)) that followed depolarizing steps in voltage-clamped, isolated mouse ventricular myocytes were examined. Depolarizing steps that produced large outward K(+) currents in these myocytes were followed by a slowly decaying inward I(tail) on repolarization to the holding potential. These currents were produced only by depolarizations: inwardly rectifying K(+) currents, I(K1), produced by steps to potentials negative to the holding potential, were not followed by I(tail). 2. For depolarizations of equal duration, the magnitude of I(tail) increased as the magnitude of outward current at the end of the depolarizing step increased. The apparent reversal potential of I(tail) was dependent upon the duration of the depolarizing step, and the reversal potential shifted to more depolarized potentials as the duration of the depolarization was increased. 3. Removal of external Na(+) and Ca(2+) had no significant effect on the magnitude or time course of I(tail). BaCl(2) (0.25 mM), which had no effect on the magnitude of outward currents, abolished I(tail) and I(K1) simultaneously. 4. Accordingly, I(tail) in mouse ventricular myocytes probably results from K(+) accumulation in a restricted extracellular space such as the transverse tubule system (t-tubules). The efflux of K(+) into the t-tubules during outward currents produced by depolarization shifts the K(+) Nernst potential (E(K)) from its 'resting' value (close to -80 mV) to more depolarized potentials. This suggests that I(tail) is produced by I(K1) in the t-tubules and is inward because of the transiently elevated K(+) concentration and depolarized value of E(K) in the t-tubules. 5. Additional evidence for the localization of I(K1) channels in the t-tubules was provided by confocal microscopy using a specific antibody against Kir2.1 in mouse ventricular myocytes.

Visualization of a water-selective pore by electron crystallography in vitreous ice.

The water-selective pathway through the aquaporin-1 membrane channel has been visualized by fitting an atomic model to a 3.7-A resolution three-dimensional density map. This map was determined by analyzing images and electron diffraction patterns of lipid-reconstituted two-dimensional crystals of aquaporin-1 preserved in vitrified buffer in the absence of any additive. The aqueous pathway is characterized by a size-selective pore that is approximately 4.0 +/- 0.5A in diameter, spans a length of approximately 18A, and bends by approximately 25 degrees as it traverses the bilayer. This narrow pore is connected by wide, funnel-shaped openings at the extracellular and cytoplasmic faces. The size-selective pore is outlined mostly by hydrophobic residues, resulting in a relatively inert pathway conducive to diffusion-limited water flow. The apex of the curved pore is close to the locations of the in-plane pseudo-2-fold symmetry axis that relates the N- and C-terminal halves and the conserved, functionally important N76 and N192 residues.

Three-dimensional fold of the human AQP1 water channel determined at 4 A resolution by electron crystallography of two-dimensional crystals embedded in ice.

Here, we present a three-dimensional (3D) density map of deglycosylated, human erythrocyte aquaporin 1 (AQP1) determined at 4 A resolution in plane and approximately 7 A resolution perpendicular to the bilayer. The map was calculated by analyzing images and electron diffraction patterns recorded from tilted (up to 60 degrees ), ice-embedded, frozen-hydrated 2D crystals of AQP1 in lipid bilayer membranes. This map significantly extends the findings related to the folding of the AQP1 polypeptide chain determined by us at a lower, 7 A by approximately 20 A, resolution. The solvent-accessible volume within a monomer has a vestibular architecture, with a narrow, approximately 6.5 A diameter constriction near the center of the bilayer, where the location of the water-selective channel is postulated to exist. The clearly resolved densities for the transmembrane helices display the protrusions expected for bulky side-chains. The density in the interior of the helix barrel (putative NPA box region) is better resolved compared to our previous map, suggesting clearer linkage to some of the helices, and it may harbor short stretches of alpha-helix. At the bilayer extremities, densities for some of the inter-helix hydrophilic loops are visible. Consistent with these observed inter-helix connections, possible models for the threading of the AQP1 polypeptide chain are presented. A preferred model is deduced that agrees with the putative locations of a group of aromatic residues in the amino acid sequence and in the 3D density map.

Effects of experimental heart failure on atrial cellular and ionic electrophysiology.

BACKGROUND: Congestive heart failure (CHF) is frequently associated with atrial fibrillation (AF), but little is known about the effects of CHF on atrial cellular electrophysiology. METHODS AND RESULTS: We studied action potential (AP) properties and ionic currents in atrial myocytes from dogs with CHF induced by ventricular pacing at 220 to 240 bpm for 5 weeks. Atrial myocytes from CHF dogs were hypertrophied (mean+/-SEM capacitance, 89+/-2 pF versus 71+/-2 pF in control, n=160 cells per group, P<0.001). CHF significantly reduced the density of L-type Ca(2+) current (I(Ca)) by approximately 30%, of transient outward K(+) current (I(to)) by approximately 50%, and of slow delayed rectifier current (I(Ks)) by approximately 30% without altering their voltage dependencies or kinetics. The inward rectifier, ultrarapid and rapid delayed rectifier, and T-type Ca(2+) currents were not altered by CHF. CHF increased transient inward Na(+)/Ca(2+) exchanger (NCX) current by approximately 45%. The AP duration of atrial myocytes was not altered by CHF at slow rates but was increased at faster rates, paralleling in vivo refractory changes. CHF created a substrate for AF, prolonging mean AF duration from 8+/-4 to 535+/-82 seconds (P<0.01). CONCLUSIONS: Experimental CHF selectively decreases atrial I(to), I(Ca), and I(Ks), increases NCX current, and leaves other currents unchanged. The cellular electrophysiological remodeling caused by CHF is quite distinct from that caused by atrial tachycardia, highlighting important differences in the cellular milieu characterizing different clinically relevant AF substrates.

Impact of the dial access drug information service on patient outcome.

OBJECTIVE: To determine the impact of a drug information service on patient outcomes. DESIGN: Prospective evaluation of patient-specific drug information requests. SETTING: Healthcare professional and consumer drug information service located at a college of pharmacy. PARTICIPANTS: Consumers and healthcare professionals of the province. INTERVENTION: Patient-specific questions received by the drug information service were reviewed and evaluated for actual patient outcome, inquirers' opinion of impact of the service with respect to patient outcome, and for objectivity and timeliness of the response. An expert panel determined whether the responses and recommendations given by the service were appropriate, determined what impact the service had on the patient, and assessed the seriousness of the inquiry. MAIN OUTCOME MEASURE: Classification of patient outcome by objective and subjective data based on predetermined desired outcomes. RESULTS: Ninety-eight and 68 patient-specific requests were received from healthcare professionals and consumers, respectively. The panel concluded that 94.9% of the healthcare requests and 98.5% of the consumer requests were answered appropriately and that the majority of the requests involved potentially serious drug-related problems. The panel also determined that 46.8% of the recommendations to healthcare professionals and 41.0% of the recommendations to consumers resulted in positive patient outcomes. The majority of the positive outcomes involved the prevention of a disease or its symptoms (professional section) and the reduction or elimination of symptoms (consumer section). CONCLUSIONS: The drug information service not only met its objectives of providing drug information in an accurate, objective, and timely manner, but was also able to provide positive patient outcomes.

Polymorphism in the packing of aquaporin-1 tetramers in 2-D crystals.

Hitherto, the packing arrangement of the aquaporin-1 (AQP1) tetramer in 2-dimensional (2-D) crystals (two-sided plane group p42(1)2) was observed to be largely similar (canonical crystal form) despite the difference in the source of the protein, the glycosylation state of the protein, the type of lipids, and the ratio of lipid to protein in the crystallization mixture. We report here our observation that the packing of AQP1 tetramers shows polymorphism in 2-D crystals generated in dioleoyl phosphatidylcholine bilayers. Apart from the canonical form, three additional allomorphs were identified. One was observed when small (0.25) lipid to protein ratio was used in the crystallization mixture while the other two were observed when the divalent cation content in the canonical crystals was modified. The various allomorphs were distinguished by different relative orientations of the AQP1 tetramer viewed in projection. The same, two-sided plane group p42(1)2 and similar unit cell dimensions were maintained in the different allomorphs as established by analysis of images of frozen-hydrated, nominally untilted crystals. Our results indicate that the interaction between the AQP1 monomers at the interface of the tetramers is flexible and is also strongly influenced by Mg(2+) ions with the cation effect materializing because of the intrinsic fluidity of the membrane.

Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation.

The rapid atrial rate during atrial fibrillation (AF) decreases the ionic current density of transient outward K+ current, L-type Ca2+ current, and Na+ current, thereby altering cardiac electrophysiology and promoting arrhythmia maintenance. To assess possible underlying changes in cardiac gene expression, we applied competitive reverse transcriptase-polymerase chain reaction to quantify mRNA concentrations in dogs subjected to 7 (group P7 dogs) or 42 (group P42 dogs) days of atrial pacing at 400 bpm and in sham controls. Rapid pacing reduced mRNA concentrations of Kv4.3 (putative gene encoding transient outward K+ current; by 60% in P7 and 74% in P42 dogs; P<0.01 and P<0.001, respectively, versus shams), the alpha1c subunit of L-type Ca2+ channels (by 57% in P7 and 72% in P42 dogs; P<0.01 versus shams for each) and the alpha subunit of cardiac Na+ channels (by 18% in P7 and 42% in P42; P=NS and P<0.01, respectively, versus shams) genes. The observed changes in ion channel mRNA concentrations paralleled previously measured changes in corresponding atrial ionic current densities. Atrial tachycardia did not affect mRNA concentrations of genes encoding delayed or Kir2.1 inward rectifier K+ currents (of which the densities are unchanged by atrial tachycardia) or of the Na+,Ca2+ exchanger. Western blot techniques were used to quantify protein expression for Kv4.3 and Na+ channel alpha subunits, which were decreased by 72% and 47%, respectively, in P42 dogs (P<0.001 versus control for each), in a manner quantitatively similar to measured changes in mRNA and currents, whereas Na+,Ca2+ exchanger protein concentration was unchanged. We conclude that chronic atrial tachycardia alters atrial ion channel gene expression, thereby altering ionic currents in a fashion that promotes the occurrence of AF. These observations provide a potential molecular basis for the self-perpetuating nature of AF.

Induction of cyclooxygenase-2 and activation of nuclear factor-kappaB in myocardium of patients with congestive heart failure.

BACKGROUND: Chronic heart failure is associated with induction of secondary inflammatory mediators, including prostanoids. The latter exert diverse functional and morphological effects on cardiac myocytes. Induction of cyclooxygenase (COX), the enzyme responsible for generating prostanoids, requires activation of nuclear factor-kappaB (NF-kappaB). The aim of the present study was to determine the expression of COX-2 and activation of NF-kappaB in the failing human heart. METHODS AND RESULTS: Myocardial tissue from 27 patients with end-stage heart failure (various etiologies: ischemic heart disease, n=16; idiopathic dilated cardiomyopathy, n=10; and valvular heart disease, n=1), 2 septic patients, and 8 normal control subjects was immunostained with antisera to COX-2 and NF-kappaB. Western blotting was performed and showed high anti-COX-2 antibody specificity and the presence of COX-2 protein in the sample tissues. In situ hybridization and immunohistochemistry showed little or no expression of COX-2 and NF-kappaB in the control hearts. In contrast, there was abundant expression of COX-2 mRNA and protein in myocytes and inflammatory cells in areas of fibrotic scar compared with regions of normal morphology in all cases of heart failure, except the cases with sepsis, which showed an abundance of COX-2 throughout the myocardium. Sites of NF-kappaB activation were associated with those of COX-2 expression. CONCLUSIONS: We demonstrate induction of COX-2 and activation of NF-kappaB in the myocardium of failing human hearts. Induction of both molecules appears to be associated with the presence of inflammation and scar formation.

Expression of endothelin-1 in pancreatic tissue of patients with chronic pancreatitis.

Chronic pancreatitis is characterized by the presence of an inflammatory infiltrate, progressive destruction of acinar cells, and fibrosis. The finding that endothelin-1, an endothelium-derived peptide with vasoconstrictive and mitogenic properties, reduces pancreatic blood flow in normal rats suggested that the peptide may be associated with the reduced pancreatic flow seen in animal models of chronic pancreatitis and in the morphological abnormalities of the disease. The aim of this study was to investigate sites of endothelin-1 expression in the pancreas of normal subjects and patients with chronic pancreatitis. The techniques of immunohistochemistry, in situ hybridization, and Northern blotting were used. Endothelin-1-like immunoreactivity was localized predominantly to islet cells both in normal subjects and in patients with chronic pancreatitis. Semi-quantitative analyses of immunostaining showed that endothelin-1-like immunoreactivity in islet cells of patients with chronic pancreatitis was greater than in normal subjects. Co-localization studies with glucagon, insulin, somatostatin, and pancreatic polypeptide showed that endothelin-1-like immunoreactivity co-exists with glucagon and insulin. There was no apparent co-existence of endothelin-1-like immunoreactivity with somatostatin or pancreatic polypeptide. Endothelin-1 mRNA was expressed in sites similar to those of the immunostaining, as well as in vascular endothelial cells. Northern blot analysis showed an increase in the expression of endothelin-1 mRNA in the patient population. There was a significant correlation between intensity of endothelin-1 immunostaining and severity of fibrosis in the patients with chronic pancreatitis. These findings suggest that an elevation in local expression of endothelin-1 may be associated with the morphological and haemodynamic changes of chronic pancreatitis.

Contamination study of multiple-dose vials.

OBJECTIVE: To document the number of opened, dated, and expired multiple-dose vials (MDVs) in patient-care areas and to determine what proportion of MDVs were contaminated with bacteria or cellular debris. DESIGN: Every tenth opened MDV (69/656) identified on the wards was collected, ensuring representation from each nursing unit. Contents were examined for contamination. SETTING: Medical-school-affiliated, tertiary care center. MAIN OUTCOME MEASURES: (1) Visual inspection for debris, medication type, location, lot number, manufacturer's expiration date, and date of opening; (2) culture in solid and broth media for bacterial growth; and (3) staining and microscopic examination for cellular constituents. RESULTS: No vials had been dated after opening and 4.6 percent were expired according to the manufacturer's expiration date. No bacterial contamination was evident; however, one vial was contaminated with red blood cells. CONCLUSIONS: Transmission of infection via contaminated MDVs has been well documented and contamination with red blood cells raises concerns about potential for transmission of bloodborne pathogens. Recommendations include dating MDVs after opening, emphasizing the need for proper aseptic technique, and discarding MDVs on the manufacture's date of expiration.

Moderate increases in peripheral blood estradiol concentration in the adult ram do not directly inhibit testosterone secretion.

Two experiments were conducted in July with adult Dorset x Leicester x Suffolk rams to determine whether increases of 150 or 300% in estradiol (E2) concentration in peripheral blood (from 6.3 +/- 0.8 pg/mL in control rams) would affect testosterone secretion directly as well as indirectly via the hypothalamic-pituitary axis. After 4 days of estradiol treatment (experiment 1) provided with subcutaneous polydimethylsiloxane implants filled with crystalline estradiol, luteinizing hormone (LH) and testosterone secretions were reduced by 50% (p < 0.05) in both groups of rams because of subtle decreases in pulse frequencies and amplitudes. Estradiol treatments were also associated with decreases in mean follicle-stimulating hormone (FSH) concentration (30-50% in both groups, p < 0.05) and increases in mean prolactin concentration (35% in low-E2 group; 105% in high-E2 group, p < 0.05), but testicular responsiveness to an LH challenge (single intravenous dose, 10 micrograms NIH-LH-S25) remained normal. When along with estradiol treatment, 10-micrograms doses LH were given every 80 min (experiment 2), testosterone secretion increased by 265% (p < 0.05) in both treated and control rams. Relative to day -1, secretion on day 4 was characterized by higher (p < 0.05) pulse frequencies and baseline concentrations and lower (p < 0.05) pulse amplitudes; values for all characteristics were similar to those for Dorset x Leicester x Suffolk rams in the breeding season. Interestingly, the decreases in mean FSH concentration brought about by estradiol and (or) LH treatments were not any greater than in experiment 1, and estradiol's ability to elevate mean prolactin concentration was blocked completely.(ABSTRACT TRUNCATED AT 250 WORDS)