Inhibition of filovirus replication by the zinc finger antiviral protein

The zinc finger antiviral protein (ZAP) was recently shown to inhibit Moloney murine leukemia virus and Sindbis virus replication. We tested whether ZAP also acts against Ebola virus (EBOV) and Marburg virus (MARV). Antiviral effects were observed after infection of cells expressing the N-terminal part of ZAP fused to the product of the zeocin resistance gene (NZAP-Zeo) as well as after infection of cells inducibly expressing full-length ZAP. EBOV was inhibited by up to 4 log units, whereas MARV was inhibited between 1 to 2 log units. The activity of ZAP was dependent on the integrity of the second and fourth zinc finger motif, as tested with cell lines expressing NZAP-Zeo mutants. Heterologous expression of EBOV- and MARV-specific sequences fused to a reporter gene suggest that ZAP specifically targets L gene sequences. The activity of NZAP-Zeo in this assay was also dependent on the integrity of the second and fourth zinc finger motif. Time-course experiments with infectious EBOV showed that ZAP reduces the level of L mRNA before the level of genomic or antigenomic RNA is affected. Transient expression of ZAP decreased the activity of an EBOV replicon system by up to 95%. This inhibitory effect could be partially compensated for by overexpression of L protein. In conclusion, the data demonstrate that ZAP exhibits antiviral activity against filoviruses, presumably by decreasing the level of viral mRNA.     

Human gene copy number spectra analysis in congenital heart malformations

The clinical significance of copy number variants (CNVs) in congenital heart disease (CHD) continues to be a challenge. Although CNVs including genes can confer disease risk, relationships between gene dosage and phenotype are still being defined. Our goal was to perform a quantitative analysis of CNVs involving 100 well-defined CHD risk genes identified through previously published human association studies in subjects with anatomically defined cardiac malformations. A novel analytical approach permitting CNV gene frequency "spectra" to be computed over prespecified regions to determine phenotype-gene dosage relationships was employed. CNVs in subjects with CHD (n = 945), subphenotyped into 40 groups and verified in accordance with the European Paediatric Cardiac Code, were compared with two control groups, a disease-free cohort (n = 2,026) and a population with coronary artery disease (n = 880). Gains (≥200 kb) and losses (≥100 kb) were determined over 100 CHD risk genes and compared using a Barnard exact test. Six subphenotypes showed significant enrichment (P ≤ 0.05), including aortic stenosis (valvar), atrioventricular canal (partial), atrioventricular septal defect with tetralogy of Fallot, subaortic stenosis, tetralogy of Fallot, and truncus arteriosus. Furthermore, CNV gene frequency spectra were enriched (P ≤ 0.05) for losses at: FKBP6, ELN, GTF2IRD1, GATA4, CRKL, TBX1, ATRX, GPC3, BCOR, ZIC3, FLNA and MID1; and gains at: PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, HRAS, GATA6 and RUNX1. Of CHD subjects, 14% had causal chromosomal abnormalities, and 4.3% had likely causal (significantly enriched), large, rare CNVs. CNV frequency spectra combined with precision phenotyping may lead to increased molecular understanding of etiologic pathways.     

Effects of IgM from rheumatic fever patients on intracellular calcium levels of neonatal rat cardiac myocytes

Rheumatic fever (RF), a potential sequela of Streptococcus pyogenes pharyngitis, sometimes results in myocarditis and heart failure. Antibodies have been implicated in the pathogenesis of RF and anti-cardiac myosin antibody levels are elevated in RF patients. Since myocarditis is associated with altered cardiomyocyte calcium transients it was of interest to determine the direct effects of RF patient antibodies on calcium transients in cultured myocytes. RF patient polyclonal IgM treatment caused increased calcium retention by neonatal rat heart cells in vitro as determined with isotopically labeled calcium. Therefore, to further characterize this finding, calcium transients were evaluated by real time fluorescence spectroscopy and deconvolution imaging. RF patient polyclonal IgM produced increased calcium retention during the relaxation stage of the contraction cycle leading to a slowing of contraction rate, disorganized calcium transients, and eventual tetany. In contrast, calcium transient studies of cardiomyocytes following treatment with monoclonal anti-myosin antibodies revealed declining intracellular calcium levels, accompanied by disorganized transients and tetany. Treatment with both antibodies led to myocyte dysfunction and these novel findings suggest a role for antibodies in the pathogenesis of the myocarditis associated with rheumatic carditis.     

A new Spionidae (Polychaeta) from North Carolina, and a redescription of Marenzelleria wireni Augener, 1913, from Spitsbergen, with a key for all species of Marenzelleria

Marenzelleria bastropi, a new species of Spionidae (Polychaeta) from the brackish water Currituck Sound, North Carolina, is described. The new species is characterized by the great number of chaetigers between the first neuro- and notopodial hooded hooks, the extension of the nuchal organ up to the end of chaetiger 2/middle of chaetiger 3 and the presence of about 60–90 branchiate chaetigers. Marenzelleria bastropi sp. nov. is closely related to M. neglecta (Sikorski and Bick, 2004) and Marenzelleria viridis (Verrill, 1873). Marenzelleria wireni Augener, 1913 is described here for the first time from western Spitsbergen. Adult specimens are investigated and compared with specimens from other areas of distribution. A key for subadult and adult specimens of all Marenzelleria species is provided.     

Atherospio guillei (Laubier and Ramos, 1974) comb. nov. (Polychaeta: Spionidae) and closest relatives

Atherospio guillei (Laubier and Ramos, 1974) comb. nov. is redescribed based on new material collected during a benthic survey in the German Bight, North Sea. Main characteristics of this species are a deeply incised prostomium, modified neurochaetae on chaetiger 5 superior to a small bundle of capillaries, branchiae fully fused to notopodial lamellae with digitiform distal process from chaetiger 7 over a limited number of succeeding chaetigers, predominately unidentate curved hooks with closely applied sheath from chaetiger 13-15 and a pygidium surrounded by several pairs of lateral cirri. Atherospio is closely related to Pygospiopsis and Pseudatherospio. Interrelationships of these genera are discussed.     

Coordination of the recruitment of the FANCD2 and PALB2 Fanconi anemia proteins by an ubiquitin signaling network

Fanconi anemia (FA) is a chromosome instability syndrome and the 20 identified FA proteins are organized into two main arms which are thought to function at distinct steps in the repair of DNA interstrand crosslinks (ICLs). These two arms include the upstream FA pathway, which culminates in the monoubiquitination of FANCD2 and FANCI, and downstream breast cancer (BRCA)-associated proteins that interact in protein complexes. How, and whether, these two groups of FA proteins are integrated is unclear. Here, we show that FANCD2 and PALB2, as indicators of the upstream and downstream arms, respectively, colocalize independently of each other in response to DNA damage induced by mitomycin C (MMC). We also show that ubiquitin chains are induced by MMC and colocalize with both FANCD2 and PALB2. Our finding that the RNF8 E3 ligase has a role in recruiting FANCD2 and PALB2 also provides support for the hypothesis that the two branches of the FA-BRCA pathway are coordinated by ubiquitin signaling. Interestingly, we find that the RNF8 partner, MDC1, as well as the ubiquitin-binding protein, RAP80, specifically recruit PALB2, while a different ubiquitin-binding protein, FAAP20, functions only in the recruitment of FANCD2. Thus, FANCD2 and PALB2 are not recruited in a single linear pathway, rather we define how their localization is coordinated and integrated by a network of ubiquitin-related proteins. We propose that such regulation may enable upstream and downstream FA proteins to act at distinct steps in the repair of ICLs.     

The calcium uptake of the rat heart sarcoplasmic reticulum is altered by dietary lipid

Summary Small amounts of dietary n-3 fatty acids can have dramatic physiological effects, including the reduction of plasma triglycerides and an elevation of cellular eicosapentanoic (EPA) and docosahexanoic acids (DHA) at the expense of arachidonic acid (AA). We investigated the effects of alterations in the fatty acid compositions of cardiac sarcoplasmic reticulum (CSR) produced by dietary manipulation on the calcium pump protein that is required for energy dependent calcium transport. CSR was isolated from rats fed menhaden oil, which is rich in n-3 fatty acids, and from control animals that were given corn oil. Relative to control membranes, those isolated from rats fed menhaden oil, had a lower content of saturated phospholipids, an increased DHA/AA ratio, and an increased ratio of n-3 to n-6 fatty acids. These changes were associated with a 30% decrease in oxalate-facilitated, ATP-dependent calcium uptake and concomitant decreased Ca-ATPase activity in the membranes from the animals fed menhaden oil. In contrast, there was no alteration in active pump sites as measured by phosphoenzyme formation. Thus, the CSR Ca-ATPase function can be altered by dietary interventions that change the composition, and possibly structure, of the phospholipid membranes thereby affecting enzyme turnover.     

Anion effects on in vitro sarcoplasmic reticulum function. Co-transport of anions with calcium

In isolated sarcoplasmic reticulum vesicles, calcium-chelating but non-calcium-precipitating dicarboxylates, such as maleate and succinate, stimulated ATP-dependent Ca2+ accumulation and its ensuring spontaneous Ca2+ accumulation and its ensuring spontaneous Ca2+ release, and Ca2+-dependent ATPase activity (Chu, A., Tate, C. A., Bick, R. J., Van Winkle, W. B., and Entman, M. L. (1983) J. Biol. Chem. 258, 1656-1664). We further examined the effect of dicarboxylates on enzyme turnover. The anionic buffer maleate enhanced the rate of rapid acyl phosphoenzyme hydrolysis compared to that in the zwitterionic buffer piperazine-N,N'-bis(2-ethanesulfonic acid) but had no effect on the phosphoenzyme formation. The presence of a calcium-precipitating anion, oxalate, or a Ca2+ ionophore, A23187, eliminated the differences observed in the phosphoenzyme decay between the two buffers, but accelerated the rate of decay. Furthermore, the catalytic activity of the purified Ca2+-dependent ATPase was not affected by maleate, whether oxalate was present or not. [14C]Succinate was transported into the sarcoplasmic reticulum in a manner which was dependent on Ca2+ transport, and occurred over a similar time course as Ca2+ accumulation/release. The net succinate uptake was equivalent to the amount of succinate-stimulated Ca2+ accumulation. Rapid efflux of both [14C]succinate and 45Ca2+ was induced by A23187, whereas the efflux induced by ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid was slower and less compared to A23187. Succinate accumulation exhibited saturation kinetics with positive cooperativity (Km congruent to 20 mM; Hill coefficient = 1.70). When maleate and succinate were both present, they were equipotent, and had an additive stimulatory effect on peak 45Ca2+ accumulation at low concentrations. Maleate was a competitive inhibitor of succinate accumulation (Ki approximately equal to 17 mM; Hill coefficient = 1.75). KCl in the presence or absence of valinomycin did not influence succinate accumulation or release. The data suggest that succinate accumulation is Ca2+-dependent, but occurs at a saturable, divalent, anion-specific site. While this carrier or channel requires Ca2+ transport, it may be controlled by additional factors as well.