A critical role for amino-terminal glutamine/asparagine repeats in the formation and propagation of a yeast prion.

The yeast [PSI+] factor propagates by a prion-like mechanism involving self-replicating Sup35p amyloids. We identified multiple Sup35p mutants that either are poorly recruited into, or cause curing of, wildtype amyloids in vivo. In vitro, these mutants showed markedly decreased rates of amyloid formation, strongly supporting the protein-only prion hypothesis. Kinetic analysis suggests that the prion state replicates by accelerating slow conformational changes rather than by providing stable nuclei. Strikingly, our mutations map exclusively within a short glutamine/asparagine-rich region of Sup35p, and all but one occur at polar residues. Even after replacement of this region with polyglutamine, Sup35p retains its ability to form amyloids. These and other considerations suggest similarities between the prion-like propagation of [PSI+] and polyglutamine-mediated pathogenesis of several neurodegenerative diseases.

The human HNRPD locus maps to 4q21 and encodes a highly conserved protein.

The hnRNP D protein interacts with nucleic acids both in vivo and in vitro. Like many other proteins that interact with RNA, it contains RBD (or "RRM") domains and arg-gly-gly (RGG) motifs. We have examined the organization and localization of the human and murine genes that encode the hnRNP D protein. Comparison of the predicted sequences of the hnRNP D proteins in human and mouse shows that they are 96.9% identical (98.9% similar). This very high level of conservation suggests a critical function for hnRNP D. Sequence analysis of the human HNRPD gene shows that the protein is encoded by eight exons and that two additional exons specify sequences in the 3' UTR. Use of two of the coding exons is determined by alternative splicing of the HNRPD mRNA. The human HNRPD gene maps to 4q21. The mouse Hnrpd gene maps to the F region of chromosome 3, which is syntenic with the human 4q21 region.

Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2.

The Drosophila brahma (brm) gene encodes an activator of homeotic genes related to the yeast chromatin remodeling factor SWI2/SNF2. Here, we report the phenotype of null and dominant-negative brm mutations. Using mosaic analysis, we found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult. A dominant-negative brm mutation was generated by replacing a conserved lysine in the ATP-binding site of the BRM protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the 2-MD BRM complex. Expression of the dominant-negative BRM protein caused peripheral nervous system defects, homeotic transformations, and decreased viability. Consistent with these findings, the BRM protein is expressed at relatively high levels in nuclei throughout the developing organism. Site-directed mutagenesis was used to investigate the functions of conserved regions of the BRM protein. Domain II is essential for brm function and is required for the assembly or stability of the BRM complex. In spite of its conservation in numerous eukaryotic regulatory proteins, the deletion of the bromodomain of the BRM protein has no discernible phenotype.

The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.

The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

Aspirin (acetylsalicylic acid) effects on behavioral thermoregulation with microwave radiation.

Aspirin is a widely used over-the-counter drug in our society which has wide therapeutic value, yet not all of the behavioral side effects have been studied. Different doses of aspirin solutions were administered (ip) prior to fixed-interval 2-min. schedules of microwave reinforcement in rats tested in a cold environment. Four Sprague-Dawley rats were conditioned to regulate their thermal environment with 5-sec. exposures of MW reinforcement. Friedman's nonparametric test showed significant differences among aspirin and saline-control doses. Post hoc sign tests showed that a moderate dose of aspirin increased operant behavior reinforced by MW radiation, yet lower and higher doses decreased and then increased the rate of responding which resulted in an inverted U-shaped trend. Possible multiple effects of aspirin in terms of its thermoregulatory as well as its pain-tolerance properties, and implications for hypothalamic "set point" are discussed.

Alcohol effects on behavioral thermoregulation with microwave radiation.

Ethanol may play an active role in modifying "set point" levels in conjunction with behavioral thermoregulation. A geometric series of doses of ethanol solutions was administered (ip) prior to fixed-interval 2-min. schedules of microwave reinforcement in rats tested in a cold environment. Four Sprague-Dawley rats were conditioned to regulate their thermal environment with 5-sec. exposures of MW reinforcement. Friedman's nonparametric test showed significant differences between ethanol doses, and Sign tests showed that moderate and high doses of ethanol suppressed operant behavior reinforced by MW radiation. Interactions between changes in "set-point" and discriminative properties of ethanol are discussed.

Capillary gas-liquid chromatography separation of phenethylamines in amphetamine-positive urine samples.

Good gas chromatography (GC) separation of molecules is essential for clean gas chromatography/mass spectrometry (GC/MS) confirmation of compounds. The trifluoro derivatives of ephedrine (E) and methamphetamine (MA) coelute on dimethyl silicone capillary columns, such as DB-1, which are most commonly used by chromatographers. Methods are described to separate E and MA to aid GC/MS confirmations of methamphetamine, ephedrine, or both E and MA together, whichever may be present in Enzyme Immunoassay (EIA)-analyzed amphetamine-positive urine samples. The use of the heptafluoro derivatives of E and MA on a DB-1 column, or the trifluoro derivatives of E and MA on a DB-17 column, is suggested for good gas chromatographic separation.

Rapid confirmation of enzyme multiplied immunoassay technique (EMIT) cocaine positive urine samples by capillary gas-liquid chromatography/nitrogen phosphorus detection (GLC/NPD).

A rapid gas-liquid chromatographic (GLC) method was developed for the confirmation of benzoylecgonine (BE) positive urine samples screened by the enzyme multiplied immunoassay technique (EMIT) assay. The procedure is performed by solvent extraction of BE from 0.1 or 0.2 mL of urine, followed by an aqueous wash of the solvent and evaporation. The dried residue was derivatized with 50 microL of pentafluoropropionic anhydride and 25 microL of pentafluoropropropanol at 90 degrees C for 15 min. The derivatizing reagents were evaporated to dryness, and the derivatized BE, and cocaine if present, were reconstituted and injected into the gas chromatograph. The column was a 15-m by 0.2-mm fused silica capillary column, coated with 0.25 micron of DB-1, terminating in a nitrogen phosphorus detector (NPD). Cocaine and the pentafluoro BE derivatives retention times were 3.2 and 2.6 min, respectively. Nalorphine was used as reference or internal standard with a retention time of 4.78 min. The complete procedure can be performed in approximately 1.5 h. The EMIT cutoff between positive and negative urine samples is 300 ng/mL of BE. The lower limit of sensitivity of this method is 25 ng of BE extracted from urine. Validation studies resulted in confirmation of 101 out of 121 EMIT cocaine positive urine samples that could not be confirmed by thin-layer chromatography (TLC). This represents 84% confirmation efficiency.

Quantitation of l-alpha-acetylmethadol and its metabolites in human serum by capillary gas-liquid chromatography and nitrogen detection.

A procedure is described for the simultaneous measurement of l-alpha-acetylmethadol and its two pharmacologically active metabolites: noracetylmethadol and dinoracetylmethadol. In the method an intramolecular conversion reaction of the two metabolites to their amide configuration is utilized. The reaction is performed while the metabolites are still in the serum. Following solvent extraction the samples are analyzed by capillary gas-liquid chromatography coupled with nitrogen detection. Quantitation is achieved by internal standardization. The lower limit of sensitivity is 5 ng/ml in serum. Absolute sensitivity is 0.1 ng for all three compounds. The advantages over other procedures are: speed due to the single extraction step; increased recovery of noracetylmethadol and dinoracetylmethadol due to decreased polarity of the amides; greater stability of the metabolites in the amide configuration; better chromatographic quantitation and separation because detector response for the amides is greater than it is for the original configuration of the metabolites and the area of the chromatographic tracing is free of interfering substances.

A rapid, quantitative GLC method for the simultaneous determination of nicotine and cotinine.

Published gas-liquid chromatographic (GLC) methods for the determination of nicotine and cotinine have proved impractical for the analysis of a large number of clinical samples. Significant improvements over other methods have been achieved, being low sample volume (0.5 mL plasma), rapid two-step extraction from plasma, no evaporation step, and good separation. The lower limits of sensitivity for nicotine and cotinine were 1 and 5 ng/mL, respectively. The method was validated by the analysis of plasma samples from cigarette-smoking volunteers. The method described permits the quick, routine determination of nicotine and cotinine in a large number of samples.

Quantitative determination of naltrexone and beta-naltrexol in human plasma using electron detection.

A gas-liquid chromatographic method is described for the determination of naltrexone and beta-naltrexol in human plasma following derivatization with pentafluoropropionic anhydride using electron capture detection. The lower sensitivity of the method for absolute standards is 5-10 pg. Following an acute 100-mg dose to a subject, peak levels of naltrexone of 15 ng/ml at 2 h and of beta-naltrexol 84 ng/ml at 4 h were observed. The levels of both compounds decreased by 24 h after the dose: naltrexone to 2.9 ng/ml and beta-naltrexol to 25 ng/ml. Following chronic administration for two weeks of 100 mg per day the peak levels of naltrexone and betanaltrexol increased to 26.9 and 131 ng/ml at 2 h, respectively, but by 24 h both compounds were at levels similar to those following a single dose. Thus no accumulation of either drug ro metabolite in the plasma was seen following chronic naltrexone administration.