The safety and tolerability of single intravenous doses of lubeluzole (Prosynap) in healthy volunteers

The safety and tolerability of single escalating doses of lubeluzole were evaluated in healthy male volunteers in 2 studies. In the first of 2 randomized, single-blind, placebo-controlled, dose-escalation studies, 6 subjects received single 30-minute infusions of 2.5, 5, and 10 mg of lubeluzole, and 2 additional subjects received placebo. In the second study 6 different subjects received a 1-hour infusion of 15 mg of lubeluzole, 5 of whom received the 20-mg dose, and 2 received 25 mg of lubeluzole. Two additional subjects received placebo. Small increases and decreases in PQ, QRS, QT, QTc, and QTm intervals were noted after infusion of all lubeluzole doses and placebo, however, these changes were within the normal ranges for these values except for the QTc for the 25-mg dose of lubeluzole. Significant prolongation of the QTc interval was observed at the end of the 1-hour infusion in both subjects receiving the 25-mg dose of lubeluzole. No clinically relevant changes in systolic time intervals, heart rate, blood pressure, and clinical laboratory values were noted in subjects receiving 2.5-25 mg of lubeluzole or placebo. Adverse experiences, predominantly lightheadedness and dizziness, were reported by subjects receiving doses of lubeluzole greater than or equal to 10 mg. Lubeluzole, administered as single intravenous doses of 2.5-15 mg, is safe and well tolerated in healthy male volunteers.     

Site-directed mutagenesis of phosphorylation sites of the branched chain alpha-ketoacid dehydrogenase complex

Regulation of the branched chain alpha-ketoacid dehydrogenase complex, the rate-limiting enzyme of branched chain amino acid catabolism, involves phosphorylation of 2 amino acid residues (site 1, serine 293; site 2, serine 303). To directly assess the roles played by these sites, site-directed mutagenesis was used to convert these serines to glutamates and/or alanines. Functional E1 heterotetramers were expressed in Escherichia coli carrying genes for E1 alpha and E1 beta under control of separate T7 promoters in a dicistronic vector. Mutation of phosphorylation site 1 serine to glutamate inactivated E1 activity, i.e. mimicked the effect of phosphorylation of site 1. Replacement of the site 1 serine with alanine greatly increased Km for the alpha-ketoacid substrate but had no effect on maximum velocity. The site 1 serine to alanine mutant was phosphorylated at site 2, but phosphorylation had no effect upon enzyme activity. Mutation of site 2 serine to either glutamate or alanine also had no effect upon enzyme activity, but phosphorylation of these proteins at site 1 inhibited enzyme activity. E1 mutated to change both phosphorylation site serines to glutamates was without enzyme activity. The binding affinity of E1 to the E2 core was not affected by mutation of the phosphorylation sites to glutamates, suggesting no gross perturbation of the association of E1 with the E2 core. The results provide direct evidence that a negative charge at phosphorylation site 1 is responsible for kinase-mediated inactivation of E1. Site 2 is silent with respect to regulation of activity by phosphorylation.     

Transcriptional regulation of cellular retinaldehyde-binding protein in the retinal pigment epithelium. A role for the photoreceptor consensus element

Cellular retinaldehyde-binding protein (CRALBP) is abundantly expressed in the retinal pigment epithelium (RPE) and Muller cells of the retina, where it is thought to function in retinoid metabolism and visual pigment regeneration. Mutations in human CRALBP that destroy retinoid binding have been linked to autosomal recessive retinitis pigmentosa. To identify the DNA elements that regulate expression of the human CRALBP gene in the RPE, transient transfection studies were carried out with three CRALBP-expressing human RPE cell culture systems. The regions from -2089 to -1539 base pairs and from -243 to +80 base pairs demonstrated positive regulatory activity. Similar activity was not observed with cultured human breast, liver, or skin cells. Since sequence analysis of the -243 to +80 region identified the presence of two photoreceptor consensus element-1 (PCE-1) sites, elements that have been implicated in photoreceptor gene regulation, the role of these sequences in RPE expression was examined. Mutation of either PCE-1 site significantly reduced reporter activity, and mutation or deletion of both sites dramatically reduced activity. Electrophoretic mobility shift analysis with RPE nuclear extracts revealed two complexes that required intact PCE-1 sites. These studies also identified two identical sequences (GCAGGA) flanking PCE-1, termed the binding CRALBP element (BCE), that are also important for complex formation. Southwestern analysis with PCE-1/BCEcontaining probes identified species with apparent masses near 90-100 and 31 kDa. These results begin to identify the regulatory regions required for RPE expression of CRALBP and suggest that PCE-1-binding factor(s) may play a role in regulating RPE as well as photoreceptor gene expression.     

Cellular retinaldehyde-binding protein ligand interactions. Gln-210 and Lys-221 are in the retinoid binding pocket

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known. We have characterized the interactions of retinoids with native bovine CRALBP, human recombinant CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or identify a Schiff base in the dark, under a variety of reducing, denaturing, and pH conditions were unsuccessful, suggesting the lack of covalent interactions between CRALBP and retinoid. Buried and solvent-exposed lysine residues were identified in bovine CRALBP by reductive methylation of the holoprotein followed by denaturation and reaction with [3H]acetic anhydride. Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteolysis and purification of modified peptides. Human rCRALBP mutants K152A, K221A, and K294A were prepared to investigate possible retinoid interactions with buried or partially buried lysines. Two other rCRALBP mutants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The structures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by UV-visible and fluorescence spectroscopy. All of the mutants bound 11-cis-retinal essentially like the wild type protein, indicating that the proteins were not grossly misfolded. Three of the mutants bound 9-cis-retinal like the wild type protein; however, Q210R and K221A bound less than stoichiometric amounts of the 9-cis-isomer and exhibited lower affinity for this retinoid relative to wild type rCRALBP. Residues Gln-210 and Lys-221 are located within a region of CRALBP exhibiting sequence homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein. The data implicate Gln-210 and Lys-221 as components of the CRALBP retinoid binding cavity and are discussed in the context of ligand interactions in structurally or functionally related proteins with known crystallographic structures.     

Structure and dynamics of the fenestrae-associated cytoskeleton of rat liver sinusoidal endothelial cells

This article describes the cytoskeleton associated with fenestrae and sieve plates of rat liver sinusoidal endothelial cells. Fenestrae control the exchange between the blood and parenchymal cells. We present evidence indicating that several agents that change the fenestrae and sieve plates also cause changes in the cytoskeleton. Cultured liver endothelial cells (LECs) were slightly fixed and treated with cytoskeleton extraction buffer. Detergent-extracted whole mounts of cultured cells were prepared for either scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Extracted cells show an integral intricate cytoskeleton; sieve plates and fenestrae are delineated by cytoskeleton elements. Fenestrae are surrounded by a filamentous, fenestrae-associated cytoskeleton with a mean filament thickness of 16 nm. Sieve plates are surrounded and delineated by microtubuli, which form a network together with additional branching cytoskeletal elements. The addition of ethanol to cultured cells enlarged the diameter for these fenestrae-associated cytoskeleton rings by 5%, whereas serotonin treatment reduced the diameter by 20%. These observations indicate that the fenestrae-associated cytoskeleton probably changes the size of fenestrae after different treatments. After treatment with cytochalasin B the number of fenestrae increased. However, cytochalasin B did not change the structure of the fenestrae-associated cytoskeleton ring, but disperses the microtubuli. In conclusion, LECs have a cytoskeleton that defines and supports sieve plates and fenestrae. Fenestrae-associated cytoskeleton is a dynamic structure and plays a role in maintaining and regulating the size of fenestrae after different treatments. Therefore, the fenestrae-associated cytoskeleton controls the important hepatic function of endothelial filtration.     

WATER-SENSITIVITY IN BARLEY II. INHIBITORS FROM BARLEY EMBRYOS

Extracts of barley embryos induced in barley grain a condition indistinguishable from water-sensitivity. Sucrose and raffinose were partly responsible but other more active and as yet unidentified compounds were also present. Inhibitory materials were found in the embryos of water-sensitive and water-insensitive barleys; barleys having a relatively high degree of water-sensitivity are particularly sensitive to further inhibitory materials such as raffinose and sucrose.     

A comparative study of empathy training with programmed instruction for lay helpers.

55 18–63 yr old lay persons were assigned to microtraining or systematic human relations empathy training (ET) or to a no-training control group. Each group, including controls, was randomly divided into subgroups for administering programmed ET to 1 subgroup in each main group. Outcome measures were levels of empathic understanding expressed in written responses to videotaped helpees. All treatment groups scored significantly higher than controls. Those subgroups, including controls, that received programmed ET via the Empathy Enhancement Tapes (R. C. Bender, 1973), scored higher than those subgroups with microtraining alone, systematic human relations training alone, or the no-tape control. Results indicate that a large-group format for teaching the skill of empathy can be an effective method of instruction and that programmed training alone and in combination with didactic methodology can have a significant impact on the enhancement of empathy. (30 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)