Interaction of scavenger receptor class B type I with peroxisomal targeting receptor Pex5p

Scavenger receptor class B type I (SR-BI) is an HDL receptor that mediates selective HDL lipid uptake. Peroxisomes play an important role in lipid metabolism and peroxisomal targeting signal type 1 (PTS1)-containing proteins are translocated to peroxisomes by the peroxisomal targeting import receptor, Pex5p. We have previously identified a PTS1 motif in the intracellular domain of rat SR-BI. Here, we examine the possible interaction between Pex5p and SR-BI. Expression of a Flag-tagged intracellular domain of SR-BI resulted in translocation to the peroxisome as demonstrated by double labeling with anti-Flag IgG and anti-catalase IgG analyzed by confocal microscopy. Immunoprecipitation experiments with anti-SR-BI antibody showed that Pex5p co-precipitated with SR-BI. However, when an antibody against Pex5p was used for immunoprecipitation, only the 57kDa, non-glycosylated form, of SR-BI co-precipitated. We conclude that the PTS1 domain of SR-BI is functional and can mediate peroxisomal interaction via Pex5p, in vitro.     

Mutants of the protein serine kinase PSKH1 disassemble the Golgi apparatus

We have dissected the molecular determinants involved in targeting the protein serine kinase PSKH1 to the endoplasmic reticulum (ER), the Golgi apparatus, and the plasma membrane (PM). Given this intracellular localization pattern, a potential role of PSKH1 in the secretory pathway was explored. The amino-terminal of PSKH1 revealed a striking similarity to the often acylated Src homology domain 4 (SH4)-harboring nonreceptor tyrosine kinases. Biochemical studies demonstrated that PSKH1 is myristoylated on glycine 2 and palmitoylated on cysteine 3. Dual amino-terminal acylation targets PSKH1 to Golgi as shown by colocalization with beta-COP and GM130, while nonpalmitoylated (myristoylated only) PSKH1 targets intracellular membranes colocalizing with protein disulphide isomerase (PDI, a marker for ER). Immunoelectron microscopy revealed that the dually acylated amino-terminal domain (in fusion with EGFP) was targeted to Golgi membranes as well as to the plasma membrane (PM), suggesting that the amino-terminal domain provides PSKH1 with membrane specificity dependent on its fatty acylation status. Subcellular fractionation by sucrose gradient analysis confirmed the impact of dual fatty acylation on endomembrane targeting, while cytosol and membrane fractioning revealed that myristoylation but not palmitoylation was required for general membrane association. A minimal region required for proper Golgi targeting of PSKH1 was identified within the first 29 amino acids. Expression of a PSKH1 mutant where the COOH-terminal kinase domain was swapped with green fluorescent protein and cysteine 3 was exchanged with serine resulted in disassembly of the Golgi apparatus as visualized by redistribution of beta-COP and GM130 to a diffuse cytoplasmic pattern, while leaving the tubulin skeleton intact. Our results suggest a structural and regulatory role of PSKH1 in maintenance of the Golgi apparatus, a key organelle within the secretory pathway.     

Identification and comparative analysis of the RpL14 gene from Takifugu rubripes

The ribosomal protein RpL14 gene has been characterized in several species, including, human, rat and fruit fly. Haploinsufficiency for the gene causes the Minute phenotype in Drosophila, and it has been proposed as a regulator in the tumorigenic pathway in human. Several features concerning the gene structure have been studied, and some of these differ between human/rat and Drosophila. To address functional and evolutionary questions about these differences we have isolated and sequenced a cDNA and a genomic clone covering the RpL14 gene from the pufferfish Takifugu rubripes (Fugu). The Fugu RpL14 gene is approximately 2 Kb, with 5 introns, and encodes a protein of 137 amino acids. The protein contains a KOW-motif and a nuclear localization signal, which are conserved among a wide range of RPL14 proteins. On the other hand, a variable amino acid (alanine) repeat observed in human is missing in Takifugu rubripes, and the protein is shorter than its mammalian counterparts. Compared with human, the RpL14 gene in Fugu contains introns localized at identical positions in the gene, and most of them are shorter. A comparison of the RpL14 gene structure from a broad range of organisms indicates that both loss and gain of introns have occurred during the evolution of the gene.     

Organization,sequence, and phylogenetic analysis of the ribosomal protein S3 gene from Drosophila virilis

Ribosomal protein S3 (RPS3) is a multifunctional ribosomal protein: it is a structural and functional component of the ribosome, and also a DNA repair enzyme involved in the DNA base excision repair pathway. Here we cloned and characterized the genomic organization of the ribosomal protein S3 gene (RpS3) homolog in Drosophila virilis. We then compared gene structure and protein sequences of RpS3 from vertebrates, invertebrates, and plants. These comparisons revealed that RpS3 genes from plants to mammals have highly conserved coding and amino acid sequences, and also protein size. Further comparisons of the protein sequences show that important domains are well conserved in both localization and sequence. In contrast, comparison of gene size and organization reveals differing patterns and levels of conservation. Whereas invertebrate RpS3 genes are small in size and gene organization is variable (from zero to four introns), vertebrates have a considerably larger (but variable) gene size and a uniform gene organization. The larger gene size in vertebrates is due to increased number and expansion of introns. Although the plant RpS3 genes are relatively small ( approximately 1.8 kb), their organization resembles that seen in vertebrates. The high conservation through different phyla may suggest that RPS3 might be under great functional constraints, both in its capacity as a component of the ribosome and as a component of a DNA repair system. Finally, electrophoretic mobility shift assays indicate that an upstream element binds a nuclear protein(s).     

Allele frequencies of apolipoprotein E gene polymorphisms in the protein coding region and promoter region (-491A/T) in a healthy Norwegian population

This study examines the distribution of apolipoprotein E (APOE) alleles in a population of healthy male and female Norwegians (n = 798) below the age of 40. The -491A/T polymorphism of the promoter region of the APOE gene was also examined. A seminested polymerase chain reaction was applied in the genotyping. The results showed that the E3 allele had the highest frequency (0.744), followed by E4 (0.198) and E2 (0.058). The APOE frequencies found in this study differ significantly from those obtained in earlier Norwegian APOE phenotypings. The allele frequencies in the -491 site of the promoter region were 0.845 for the A allele and 0.155 for the T allele. The genotype frequency was highest for AA (0.707), followed by AT (0.277) and TT (0.016). Moreover, the A allele was in linkage disequilibrium to E4.     

AII (Rod) amacrine cells form a network of electrically coupled interneurons in the mammalian retina

AII (rod) amacrine cells in the mammalian retina are reciprocally connected via gap junctions, but there is no physiological evidence that demonstrates a proposed function as electrical synapses. In whole-cell recordings from pairs of AII amacrine cells in a slice preparation of the rat retina, bidirectional, nonrectifying electrical coupling was observed in all pairs with overlapping dendritic trees (average conductance approximately 700 pS). Coupling displayed characteristics of a low-pass filter, with no evidence for amplification of spike-evoked electrical postsynaptic potentials by active conductances. Coincidence detection, as well as precise temporal synchronization of subthreshold membrane potential oscillations and TTX-sensitive spiking, was commonly observed. These results indicate a unique mode of operation and integrative capability of the network of AII amacrine cells.     

The effect of brominated flame retardants on neurotransmitter uptake into rat brain synaptosomes and vesicles

The environmental levels of brominated flame retardants (BFRs) are increasing, but little is known about their toxic effects. In this paper, we show that some of the most important BFRs in commercial use today, have a neurotoxicological potential. Hexabromocyclododecane (HBCD) and tetrabromobisphenol-A (TBBPA) inhibit plasma membrane uptake of the neurotransmitters dopamine, glutamate and gamma-amino-n-butyric acid (GABA) at a concentration level similar to what previously found for polychlorinated biphenyls (PCBs) and even for ecstasy. The IC(50) value for HBCD on dopamine uptake was 4 microM, and the IC(50) values for TBBPA were 9, 6 and 16 microM for dopamine, glutamate and GABA, respectively. HBCD also inhibited glutamate uptake at low concentrations, but never achieved more than 50% inhibition. The inhibition was primarily due to their effect on the membrane potential, measured by the membrane potential marker tetraphenylphosphonium bromide (TPP(+)). Other brominated flame retardants such as octaBDE and decaBDE did not have any effects on uptake. TBBPA, HBCD and even the pentabrominated diphenylether mixture (pentaBDE, DE-71, Great Lakes) also inhibited the vesicular uptake of dopamine with an IC(50) value of 3, 3 and 8 microM, respectively. The neurotoxicological consequences of these findings for environmental contaminants such as BFRs and PCBs are discussed.     

Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.     

Sprint Conditioning of Junior Soccer Players: Effects of Training Intensity and Technique Supervision

The aims of the present study were to compare the effects of 1) training at 90 and 100% sprint velocity and 2) supervised versus unsupervised sprint training on soccer-specific physical performance in junior soccer players. Young, male soccer players (17 ±1 yr, 71 ±10 kg, 180 ±6 cm) were randomly assigned to four different treatment conditions over a 7-week intervention period. A control group (CON, n=9) completed regular soccer training according to their teams’ original training plans. Three training groups performed a weekly repeated-sprint training session in addition to their regular soccer training sessions performed at A) 100% intensity without supervision (100UNSUP, n=13), B) 90% of maximal sprint velocity with supervision (90SUP, n=10) or C) 90% of maximal sprint velocity without supervision (90UNSUP, n=13). Repetitions x distance for the sprint-training sessions were 15x20 m for 100UNSUP and 30x20 m for 90SUP and 90UNSUP. Single-sprint performance (best time from 15x20 m sprints), repeated-sprint performance (mean time over 15x20 m sprints), countermovement jump and Yo-Yo Intermittent Recovery Level 1 (Yo-Yo IR1) were assessed during pre-training and post-training tests. No significant differences in performance outcomes were observed across groups. 90SUP improved Yo-Yo IR1 by a moderate margin compared to controls, while all other effect magnitudes were trivial or small. In conclusion, neither weekly sprint training at 90 or 100% velocity, nor supervised sprint training enhanced soccer-specific physical performance in junior soccer players.