Peroxiredoxin IV is an endoplasmic reticulum-localized enzyme forming oligomeric complexes in human cells

The peroxiredoxins are a ubiquitous family of proteins involved in protection against oxidative stress through the detoxification of cellular peroxides. In addition, the typical 2-Cys peroxiredoxins function in signalling of peroxide stress and as molecular chaperones, functions that are influenced by their oligomeric state. Of the human peroxiredoxins, Prx IV (peroxiredoxin IV) is unique in possessing an N-terminal signal peptide believed to allow secretion from the cell. Here, we present a characterization of Prx IV in human cells demonstrating that it is actually retained within the ER (endoplasmic reticulum). Stable knockdown of Prx IV expression led to detrimental effects on the viability of human HT1080 cells following treatment with exogenous H2O2. However, these effects were not consistent with a dose-dependent correlation between Prx IV expression and peroxide tolerance. Moreover, modulation of Prx IV expression showed no obvious effect on ER-associated stress, redox conditions or H2O2 turnover. Subsequent investigation demonstrated that Prx IV forms complex structures within the ER, consistent with the formation of homodecamers. Furthermore, Prx IV oligomeric interactions are stabilized by additional non-catalytic disulfide bonds, indicative of a primary role other than peroxide elimination.     

Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria)

Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ss-tubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent "robust" and "complex" clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils.     

Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase Cgamma1, Grb2, and Src family kinases

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.     

Perkinsus olseni detected in Vietnamese aquacultured reef clams Tridacna crocea imported to the USA, following a mortality event

Morbidity and mortality were observed in a group of 30 reef clams Tridacna crocea that were imported to Florida, USA, from a Vietnamese culture facility and held in research facility aquaria. Clinical signs included an incompletely extended mantle, slow mantle responses to stimuli, and sloughing of byssal tissue beginning 2 to 5 d prior to death. Necropsy findings included emaciation, visceral mass edema, and rare multifocal 1 mm off-white to light-tan gill nodules. Histopathology revealed marked inflammation and necrosis within the visceral mass and gills, with interstitial edema and atrophy of glandular, gonadal, and muscular tissues. Inflamed tissues contained large numbers of 10 to 15 microm extracellular round organisms consistent with Perkinsus sp. trophozoites. The organisms often formed clusters of 1 to 4 cells and were surrounded by a 1 to 3 microm rim of eosinophilic material variably forming a radiating corona pattern and by 3 to 4 host hemocytes with dense round nuclei. Polymerase chain reaction assays indicated the presence of Perkinsus sp. DNA in these animals, and species-specific assays indicated the presence of P. olseni, and possibly other Perkinsus spp., but not P. marinus. Identification of Perkinsus spp. other than P. marinus in T. crocea imported from Vietnam confirms that importation of untested and unquarantined ornamental reef clams has possibly allowed incursion of P. olseni into the USA.     

Enzyme microgels in packed-bed bioreactors with downstream amperometric detection using microfabricated interdigitated microsensor electrode arrays.

In this article, we describe the use of pH- responsive hydrogels as matrices for the immobilization of two enzymes, glucose oxidase (GOx) and glutamate oxidase (GlutOx). Spherical hydrogel beads were prepared by inverse suspension polymerization and the enzymes were immobilized by either physical entrapment or covalent immobilization within or on the hydrogel surface. Packed-bed bioreactors were prepared containing the bioactive hydrogels and these incorporated into flow injection (FI) systems for the quantitation of glucose and monosodium glutamate (MSG) respectively. The FI amperometric detector comprised a microfabricated interdigitated array within a thin-layer flow cell. For the FI manifold incorporating immobilized GOx, glucose response curves were found to be linear over the concentration range 1.8-280 mg dL(-1) (0.1-15.5 mM) with a detection limit of 1.4 mg dL(-1) (0.08 mM). Up to 20 samples can be manually analyzed per hour, with the hydrogel-GOx bioreactor exhibiting good within-day (0.19%) precision. The optimized FI manifold for MSG quantitation yielded a linear response range of up to 135 mg dL(-1) (8 mM) with a detection limit of 3.38 mg dL(-1) (0.2 mM) and a throughput of 30 samples h(-1). Analysis of commercially produced soup samples gave a within-day precision of 3.6%. Bioreactors containing these two physically entrapped enzymes retained > 60% of their initial activities after a storage period of up to 1 year.     

Chimeric Human Immunodeficiency Virus Type 1 Containing Murine Leukemia Virus Matrix Assembles in Murine Cells

Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of the precursor is mediated by the amino-terminal region of polyprotein. To compensate for the assembly block, we substituted the murine leukemia virus matrix coding sequences into an infectious HIV-1 clone. Transfection of murine fibroblasts expressing cyclin T1 with the chimeric proviruses resulted in viruses that were efficiently assembled and released. Chimeric viruses, in which the cytoplasmic tail of the transmembrane subunit, gp41, was truncated to prevent potential interference between the envelope glycoprotein and the heterologous matrix, could infect human and murine cells. They failed to further replicate in the murine cells, but replicated with delayed kinetics in human MT-4 cells. These findings may be useful for establishing a murine model for HIV-1 replication.