In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.     

Chloroplasts as source and target of cellular redox regulation: a discussion on chloroplast redox signals in the context of plant physiology

During the evolution of plants, chloroplasts have lost the exclusive genetic control over redox regulation and antioxidant gene expression. Together with many other genes, all genes encoding antioxidant enzymes and enzymes involved in the biosynthesis of low molecular weight antioxidants were transferred to the nucleus. On the other hand, photosynthesis bears a high risk for photo-oxidative damage. Concomitantly, an intricate network for mutual regulation by anthero- and retrograde signals has emerged to co-ordinate the activities of the different genetic and metabolic compartments. A major focus of recent research in chloroplast regulation addressed the mechanisms of redox sensing and signal transmission, the identification of regulatory targets, and the understanding of adaptation mechanisms. In addition to redox signals communicated through signalling cascades also used in pathogen and wounding responses, specific chloroplast signals control nuclear gene expression. Signalling pathways are triggered by the redox state of the plastoquinone pool, the thioredoxin system, and the acceptor availability at photosystem I, in addition to control by oxolipins, tetrapyrroles, carbohydrates, and abscisic acid. The signalling function is discussed in the context of regulatory circuitries that control the expression of antioxidant enzymes and redox modulators, demonstrating the principal role of chloroplasts as the source and target of redox regulation.     

Genetic analysis of thirty-three platelet polypeptides detected in two-dimensional polyacrylamide gels.

Two-dimensional gel electrophoresis followed by silver-staining was utilized to visualize platelet polypeptides for genetic analysis. A subset of 33 polypeptides that were most suited for scoring was selected. Families consisting of father-mother-child trios were studied. Thirty-six polypeptides of a total of 1,413 scored in children's gels exhibited the combination of a normal and a variant polypeptide. The observed index of heterozygosity of 2.55% is comparable to our previously reported findings for red cell proteins.     

Genomic effects of IFN-beta in multiple sclerosis patients

The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-beta-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 micro g of IFN-beta-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-beta-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1-4 h of IFN-beta treatment. The mRNAs for several genes involved in IFN-beta signaling, such as IFN-alpha/beta receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9-27), IFN-induced transmembrane protein 2 (1-8D), beta(2)-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-beta treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.     

The function of the chloroplast 2-cysteine peroxiredoxin in peroxide detoxification and its regulation

The Arabidopsis genome contains nine open reading frames with homology to members of the peroxiredoxin (prx) family: one 1-Cys-prx, two 2-Cys-prx, five type II-prx, and one peroxiredoxin Q. The function of the peroxiredoxins in plant metabolism is only slowly emerging. They are assumed to reduce toxic peroxides to their corresponding alcohols with a rather broad substrate specificity. The 2-Cys peroxiredoxins (2-CP) were recently identified as members of the antioxidant defence system of chloroplasts. Knock-out mutants of Synechocystis and antisense mutants of Arabidopsis have provided insight into the function of 2-CPs in the photosynthetic antioxidant network. This review summarizes present knowledge on the enzymatic mechanism, the physiological context and the genetic regulation of the 2-CPs in plants and cyanobacteria. In addition, an extrapolation on the metabolic role of the chloroplast 2-CP is attempted based on the molecular features of 2-CPs from other organisms.     

Effects of an Ala54Thr polymorphism in the intestinal fatty acid-binding protein on responses to dietary fat in humans

A polymorphism in FABP2 that results in an alanine-to-threonine substitution at amino acid 54 of the intestinal fatty acid-binding protein (IFABP) is associated with insulin resistance in Pima Indians. In vitro, the threonine form (Thr54) has a higher binding affinity for long-chain fatty acids than does the alanine form (Ala54). We tested whether this polymorphism affected metabolic responses to dietary fat, in vivo. Eighteen healthy Pima Indians, half homozygous for the Thr54 form of IFABP and half homozygous for the Ala54 form, were studied. The groups were matched for sex, age, and body mass index. Plasma triglyceride, nonesterified fatty acid (NEFA), glucose, and insulin responses were measured after a mixed meal (35% of daily energy requirements, 50 g of fat) and after a high fat challenge (1362 kcal, 129 g of fat). NEFA concentrations were approximately 15% higher after the mixed meal and peaked earlier and were approximately 20% higher at 7 h in response to the high fat test meal in Thr54 homozygotes compared with Ala54 homozygotes. Insulin responses to the test meals tended to be higher in Thr54 homozygotes, but glucose and triglyceride responses were not different.The results of this study suggest that the Thr54 form of IFABP is associated with higher and prolonged NEFA responses to dietary fat in vivo. Higher NEFA concentrations may contribute to insulin resistance and hyperinsulinemia in individuals with this allele.     

Analysis of Staphylococcal Enterotoxin B by the Polyacrylamide Electrophoresis Technique

The electrophoretic mobility of enterotoxin B was investigated through the use of the disc electrophoresis technique. Ideal patterns were developed with a 7.5% acrylamide gel system (pH 4.3). The toxin can be separated and identified from other complex proteins such as serum or suspect samples of foods by this technique. The technique can be used as an assay method for the toxin as well as to elucidate physical changes in the toxin due to temperature. The method should not be considered exclusive for enterotoxin B.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.