A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA

Amyloid fibrils from a patient with diffuse amyloid disease are dissociated in 6 m guanidine hydrochloride and fractionated by gel chromatography. Two major components are separated on Sepharose 6B. Both proteins are characterized by chromatography, immunodiffusion, discontinuous gel electrophoresis, amino acid tryptic peptide mapping and amino acid sequence analysis. The smaller of the two components is typical of the known protein AA by size (8400 daltons), amino acid composition and a 30-residue N-terminal sequence. The larger of the components (25,000 daltons) undergoes electrophoresis as a single band and appears unaffected by thiol reduction. It differs from protein AA in amino acid content and by its tryptic peptide map, although it contains an N-terminal amino acid sequence identical to protein AA when carried to 20 residues. Treatment of this larger component by mild acid hydrolysis results in the release of the 8400-dalton protein AA. Fractionation after guanidine hydrochloride treatment of this particular amyloid fibril preparation is compared to the fractionation of a typical secondary amyloid preparation that contains only protein AA as the major component. The origin and relationship of the 8,400- and 25,000-dalton protein components is discussed.     

THE EFFECT OF ENZYME PURITY ON THE KINETICS OF TRYPTIC HYDROLYSIS

The rates of digestion of keratose have been determined with three commercial enzymes, ranging widely in strength. It has been found that the weaker the enzyme preparation, the more nearly does the course of the hydrolysis conform to that of a reaction of the first order. This has been explained on the assumption that in solution an equilibrium exists between active enzyme, and enzyme combined with inert material. In very impure enzyme preparations, the large quantities of combined enzyme act as a reservoir for active enzyme, maintaining a constant concentration of active enzyme during the course of the digestion.     

The reactions of the phosphorylated pathway of L-serine biosynthesis: thermodynamic relationships in rabbit liver in vivo

The thermodynamic relationships among the reactions of the phosphorylated pathway of L-serine biosynthesis have been determined in rabbit liver in vivo in different dietary states. The mass action ratios of the reactions involved were calculated from the concentrations of appropriate metabolites in freeze-clamped liver and compared with the equilibrium constants of the same reactions previously determined under physiological conditions. Toward this goal, a new, highly specific enzymatic assay for L-phosphoserine was developed to allow the accurate measurement of this intermediate in biological material. The level of L-phosphoserine, the immediate precursor of L-serine, varied significantly with diet, being 0.81, 0.38, and 0.21 mumol/g wet wt in the fed, and 24 h and 48 h fasted states, respectively. The tissue content of L-phosphoserine was also sensitive to anoxia, falling almost fivefold within 5 min after the liver was removed. Values of for the combined reactions of the first two steps of the pathway of L-serine biosynthesis [D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) and L-phosphoserine aminotransferase (EC 2.6.1.52)] in livers from animals in different dietary states were calculated to be 1.2 X 10(-4) (fed), 1.4 X 10(-4) (24 h starved), and 0.70 X 10(-4) (48 h starved), all being very close to the value of the combined equilibrium constant of the same reactions (2.44 X 10(-4). Even when there were major changes in the individual components of, such as a fivefold drop in L-phosphoserine and a sevenfold fall in alpha-ketoglutarate following 5 min of anoxia, remained relatively unchanged (2.7 X 10(-4). Thus, it has been concluded that, in rabbit liver under most normal conditions, the combined reactions of D-3-phosphoglycerate dehydrogenase and L-phosphoserine aminotransferase remain very near equilibrium, and that almost all of the disequilibrium of the pathway, amounting to a delta G of -5.5 kcal/mol in the fed state, is at the last step, the L-phosphoserine phosphatase reaction (EC 3.1.3.3).     

In vitro toxicology of fumonisins and the mechanistic implications

The effects of fumonisins B₁FB₁, B₂(FB{2}), and the backbone of fumonisin B₁ remaining after hydrolysis of the tricarballylic groups with base (HFB₁) on sphingolipid biosynthesis were studied in both primary rat hepatocytes and pig kidney epithelial cells (LLC-PK₁). Fumonisins were potent inhibitors of sphingolipid biosynthesis in hepatocytes (IC₅₀ of FB₁=0.1 M), but overt toxicity was not observed. In renal cells, fumonisins also inhibited sphingosine biosynthesis (IC₅₀ for FB₁=35 M), and caused decreased cell proliferation as well. Higher doses (70 M) killed renal cells after exposure for 3 days. The inhibition of de novo sphingolipid biosynthesis was specific, and appeared to be at the site of ceramide synthase, which catalyzes the formation of dihydroceramide or ceramide by the addition of the amide-linked fatty acid to sphinganine or sphingosine. These results may account for the ability of fumonisins to cause equine leucoencephalomalacia and to promote tumor formation.     

Odorant-specific requirements for arrestin function in Drosophila olfaction

The ability to modulate olfactory sensitivity is necessary to detect chemical gradients and discriminate among a multitude of odor stimuli. Desensitization of odorant receptors has been postulated to occur when arrestins prevent the activation of downstream second messengers. A paucity of in vivo data on olfactory desensitization prompts use of Drosophila melanogaster genetics to investigate arrestins' role in regulating olfactory signaling pathways. Physiological analysis of peripheral olfactory sensitivity reveals decreased responsiveness to a host of chemically distinct odorants in flies deficient for arrestin1 (arr1), arrestin2 (arr2), or both. These phenotypes are manifest in odorant- and dose- dependent fashions. Additionally, mutants display altered adaptive properties under a prolonged exposure paradigm. Behaviorally, arr1 mutants are impaired in olfactory-based orientation towards attractive odor sources. As the olfactory deficits vary according to chemical identity and concentration, they indicate that a spectrum of arrestin activity is essential for odor processing depending upon the particular olfactory pathway involved. Arrestin mutant phenotypes are hypothesized to be a consequence of down-regulation of olfactory signaling to avoid cellular excitotoxicity. Importantly, phenotypic rescue of olfactory defects in arr1(1) mutants is achieved through transgenic expression of wild-type arr1. Taken together, these data clearly indicate that arrestins are required in a stimulus-specific manner for wild type olfactory function and add another level of complexity to peripheral odor coding mechanisms that ultimately impact olfactory behavior.     

Development of a novel automated ion channel recording method using "inside-out" whole-cell membranes

Efforts to develop novel methods for recording from ion channels have been receiving increased attention in recent years. In this study, the authors report a unique "inside-out" whole-cell configuration of patch-clamp recording that has been developed. This method entails adding cells into a standard patch pipette and, with positive pressure, obtaining a gigaseal recording from a cell at the inside tip of the electrode. In this configuration, the cell may be moved through the air, first rupturing part of the cellular membrane and enabling bath access to the intracellular side of the membrane, and then into a series of wells containing differing solutions, enabling robotic control of all the steps in an experiment. The robotic system developed here fully automates the electrophysiological experiments, including gigaseal formation, obtaining whole-cell configuration, data acquisition, and drug application. Proof-of-principle experiments consisting of application of intracellularly acting potassium channel blockers to K+ channel cell lines resulted in a very rapid block, as well as block reversal, of the current. This technique allows compound application directly to the intracellular side of ion channels and enables the dissociation of compound in activities due to cellular barrier limitations. This technique should allow for parallel implementation of recording pipettes and the future development of larger array-based screening methods.     

What Are the Roles and Responsibilities of the Media in Disseminating Health Information?

BACKGROUND TO THE DEBATE: In December 2004 three news stories in the popular press suggested that the side effects of single-dose nevirapine, which has been proven to prevent mother-to-child transmission of HIV, had been covered up. Many HIV experts believed that the stories were unwarranted and that they would undermine use of the drug, leading to a rise in neonatal HIV infection. The controversy surrounding these stories prompted the PLoS Medicine editors to ask health journalists, and others with an interest in media reporting of health, to share their views on the roles and responsibilities of the media in disseminating health information.