A fluorescent sphingolipid binding domain peptide probe interacts with sphingolipids and cholesterol-dependent raft domains

We have designed a tagged probe [sphingolipid binding domain (SBD)] to facilitate the tracking of intracellular movements of sphingolipids in living neuronal cells. SBD is a small peptide consisting of the SBD of the amyloid precursor protein. It can be conjugated to a fluorophore of choice and exogenously applied to cells, thus allowing for in vivo imaging. Here, we present evidence to describe the characteristics of the SBD association with the plasma membrane. Our experiments demonstrate that SBD binds to isolated raft fractions from human neuroblastomas and insect neuronal cells. In protein-lipid overlay experiments, SBD interacts with a subset of glycosphingolipids and sphingomyelin, consistent with its raft association in neurons. We also provide evidence that SBD is taken up by neuronal cells in a cholesterol- and sphingolipid-dependent manner via detergent-resistant microdomains. Furthermore, using fluorescence correlation spectroscopy to assay the mobility of SBD in live cells, we show that SBD's behavior at the plasma membrane is similar to that of the previously described raft marker cholera toxin B, displaying both a fast and a slow component. Our data suggest that fluorescently tagged SBD can be used to investigate the dynamic nature of glycosphingolipid-rich detergent-resistant microdomains that are cholesterol-dependent.     

Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain

Chondroitin sulphate proteoglycans (CSPGs) are axon growth inhibitory molecules present in the glial scar that play a part in regeneration failure after damage to the CNS and which restrict CNS plasticity. Removal of chondroitin sulphate glycosaminoglycan (GAG) chains with chondroitinase-ABC (chABC) in models of CNS injury promotes both axon regeneration and plasticity. We have analysed the immediate and long-term effects of a single injection of chABC on CSPGs, GAGs and axon regeneration. We made unilateral nigrostriatal lesions in adult rats accompanied by an adjacent infusion of either chABC or a bacterial-derived control enzyme (penicillinase). Within 24 h of chABC treatment there was digestion of GAGs, including hyaluronan, and a reduction in neurocan in an area extending 1.5 mm around the injection site. Around 50% of GAG is inaccessible to chABC digestion, even in tissue digested in vitro, which probably represents intracellular stores. In control penicillinase treated animals, total GAGs recovered from the lesioned brains were up-regulated by 4-fold 7 days after injury and gradually decreased to normal at 28 days post-lesion. In chondroitinase-treated animals, the total GAG remained at low level throughout the 28-day experimental period. This suggests the persistence of active chABC for at least 10 days after injection which is able to digest CSPGs released from cells during this time. This was confirmed by immunological detection of enzyme for 10 days and by retrieval of active enzyme from the brain at 10 days after injection. Our results suggest that a single injection of chABC can produce an environment conducive to CNS repair for over 10 days.     

Contrasting responses of lymphoid progenitors to canonical and noncanonical Wnt signals

The Wnt family of secreted glycoproteins has been implicated in many aspects of development, but its contribution to blood cell formation is controversial. We overexpressed Wnt3a, Wnt5a, and Dickkopf 1 in stromal cells from osteopetrotic mice and used them in coculture experiments with highly enriched stem and progenitor cells. The objective was to learn whether and how particular stages of B lymphopoiesis are responsive to these Wnt family ligands. We found that canonical Wnt signaling, through Wnt3a, inhibited B and plasmacytoid dendritic cell, but not conventional dendritic cell development. Wnt5a, which can oppose canonical signaling or act through a different pathway, increased B lymphopoiesis. Responsiveness to both Wnt ligands diminished with time in culture and stage of development. That is, only hematopoietic stem cells and very primitive progenitors were affected. Although Wnt3a promoted retention of hematopoietic stem cell markers, cell yields and dye dilution experiments indicated it was not a growth stimulus. Other results suggest that lineage instability results from canonical Wnt signaling. Lymphoid progenitors rapidly down-regulated RAG-1, and some acquired stem cell-staining characteristics as well as myeloid and erythroid potential when exposed to Wnt3a-producing stromal cells. We conclude that at least two Wnt ligands can differentially regulate early events in B lymphopoiesis, affecting entry and progression in distinct differentiation lineages.     

Murine coronaviruses encoding nsp2 at different genomic loci have altered replication, protein expression, and localization

Partial or complete deletion of several coronavirus nonstructural proteins (nsps), including open reading frame 1a (ORF1a)-encoded nsp2, results in viable mutant proteins with specific replication defects. It is not known whether expression of nsps from alternate locations in the genome can complement replication defects. In this report, we show that the murine hepatitis virus nsp2 sequence was tolerated in ORF1b with an in-frame insertion between nsp13 and nsp14 and in place of ORF4. Alternate encoding or duplication of the nsp2 gene sequence resulted in differences in nsp2 expression, processing, and localization, was neutral or detrimental to replication, and did not complement an ORF1a Δnsp2 replication defect. The results suggest that wild-type genomic organization and expression of nsps are required for optimal replication.     

Mutations in the amino terminus of foamy virus Gag disrupt morphology and infectivity but do not target assembly

Foamy viruses (FVs) assemble using pathways distinct from those of orthoretroviruses. FV capsid assembly takes place near the host microtubule-organizing center (MTOC). Assembled capsids then migrate by an unknown mechanism to the trans-Golgi network to colocalize with the FV glycoprotein, Env. Interaction with Env is required for FV capsid egress from cells; the amino terminus of FV Gag contains a cytoplasmic targeting/retention signal that is responsible for targeting assembly to the MTOC. A mutant Gag was constructed by addition of a myristylation (M) signal in an attempt to target assembly to the plasma membrane and potentially overcome the dependence upon Env for budding (S. W. Eastman and M. L. Linial, J. Virol. 75:6857-6864, 2001). Using this and additional mutants, we now show that assembly is not redirected to the plasma membrane. Addition of an M signal leads to gross morphological defects. The aberrant particles still assemble near the MTOC but do not produce infectious virus. Although extracellular Gag can be detected in a pelletable form in the absence of Env, the mutant particles contain very little genomic RNA and are less dense. Our analyses indicate that the amino terminus of Gag contains an Env interaction domain that is critical for bona fide egress of assembled capsids.     

Differential requirements of the C terminus of Nbs1 in suppressing adenovirus DNA replication and promoting concatemer formation

Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.     

Large inverted repeats in the vicinity of a single double-strand break strongly affect repair in yeast diploids lacking Rad51

DNA double-strand breaks (DSBs) are critical lesions that can lead to cell death or chromosomal rearrangements. Rad51 is necessary for most mitotic and meiotic DSB repair events, although a number of RAD51-independent pathways exist. Previously, we described DSB repair in rad51Delta yeast diploids that was stimulated by a DNA region termed "facilitator of break-induced replication" (FBI) located approximately 30kb from the site of an HO-induced DSB. Here, we demonstrate that FBI is a large inverted DNA repeat that channels the repair of DSBs into the single-strand annealing-gross chromosomal rearrangements (SSA-GCR) pathway. Further, analysis of DSB repair in rad54Delta cells allowed us to propose that the SSA-GCR repair pathway is suppressed in the presence of Rad51p. Therefore, an additional role of Rad51 might be to protect eukaryotic genomes from instabilities by preventing chromosomal rearrangements.     

Nucleophosmin serves as a rate-limiting nuclear export chaperone for the Mammalian ribosome

Nucleophosmin (NPM) (B23) is an essential protein in mouse development and cell growth; however, it has been assigned numerous roles in very diverse cellular processes. Here, we present a unified mechanism for NPM's role in cell growth; NPM directs the nuclear export of both 40S and 60S ribosomal subunits. NPM interacts with rRNA and large and small ribosomal subunit proteins and also colocalizes with large and small ribosomal subunit proteins in the nucleolus, nucleus, and cytoplasm. The transduction of NPM shuttling-defective mutants or the loss of Npm1 inhibited the nuclear export of both the 40S and 60S ribosomal subunits, reduced the available pool of cytoplasmic polysomes, and diminished overall protein synthesis without affecting rRNA processing or ribosome assembly. While the inhibition of NPM shuttling can block cellular proliferation, the dramatic effects on ribosome export occur prior to cell cycle inhibition. Modest increases in NPM expression amplified the export of newly synthesized rRNAs, resulting in increased rates of protein synthesis and indicating that NPM is rate limiting in this pathway. These results support the idea that NPM-regulated ribosome export is a fundamental process in cell growth.     

Analysis of TaALMT1 traces the transmission of aluminum resistance in cultivated common wheat (Triticum aestivum L.)

Allele diversities of four markers specific to intron three, exon four and promoter regions of the aluminum (Al) resistance gene of wheat (Triticum aestivum L.) TaALMT1 were compared in 179 common wheat cultivars used in international wheat breeding programs. In wheat cultivars released during the last 93 years, six different promoter types were identified on the basis of allele size. A previous study showed that Al resistance was not associated with a particular coding allele for TaALMT1 but was correlated with blocks of repeated sequence upstream of the coding sequence. We verified the linkage between these promoter alleles and Al resistance in three doubled haploid and one intercross populations segregating for Al resistance. Molecular and pedigree analysis suggest that Al resistance in modern wheat germplasm is derived from several independent sources. Analysis of a population of 278 landraces and subspecies of wheat showed that most of the promoter alleles associated with Al resistance pre-existed in Europe, the Middle East and Asia prior to dispersal of cultivated germplasm around the world. Furthermore, several new promoter alleles were identified among the landraces surveyed. The TaALMT1 promoter alleles found within the spelt wheats were consistent with the hypothesis that these spelts arose on several independent occasions from hybridisations between non-free-threshing tetraploid wheats and Al-resistant hexaploid bread wheats. The strong correlation between Al resistance and Al-stimulated malate efflux from the root apices of 49 diverse wheat genotypes examined was consistent with the previous finding that Al resistance in wheat is conditioned primarily by malate efflux. These results demonstrate that the markers based on intron, exon and promoter regions of TaALMT1 can trace the inheritance of the Al resistance locus within wheat pedigrees and track Al resistance in breeding programmes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of horse spleen apoferritin reactive lysines by MALDI-TOF mass spectrometry combined with enzymatic digestion

Ferritins are a class of iron storage protein spheres found mainly in the liver and spleen, which have attracted many research interests due to their unique structural features and biological properties. Recently, ferritin and apoferritin (ferritin devoid of the iron core), have been employed as chemically addressable nanoscale building blocks for functional materials development. However, the reactive residues of apoferritin or ferritin have never been specified and it is still unclear about the chemoselectivity of apoferritin towards different kinds of bioconjugation reagents. In this work, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry combined with enzymatic digestion analysis was used to identify the reactive lysine residues of horse spleen apoferritin when conjugated with N-hydroxysuccinimide reagents. The result demonstrated that among all the lysine residues, K97, K83, K104, K67 and K143 are the reactive ones that can be addressed.