A novel mechanism for the initiation of Tacaribe arenavirus genome replication.

The ends of arenavirus genome and antigenome RNAs are highly conserved and where determined directly, always contain a 3' G (referred to as position +1). However, primers extended to the 5' ends of Tacaribe virus genomes and antigenomes extend to position -1. When genomes and antigenomes are annealed either inter or intramolecularly and treated with RNase A or T1, there appears to be a single unpaired G at the 5' ends of the hybrids. A single extra G is also found by cloning the 5' ends of S antigenomes, and studies with capping enzyme detect (p)ppG at the 5' ends of genome and antigenome chains. A model is proposed in which genome replication initiates with pppGpC to create the nontemplated extra G. In contrast, the nontemplated bases at the 5' ends of the N mRNAs, which extend to positions -1 to -5, were found to be capped and also heterogeneous in sequence.     

Retinoic acid decreases retinoic acid and triiodothyronine nuclear receptor expression in the liver of hyperthyroidic rats.

Retinoic acid (RA) and triiodothyronine (T3) exert many of their actions by binding to specific nuclear receptors (respectively, RA receptor (RAR) and T3) receptor (TR) belonging to a 'superfamily' of receptors. Some heterologous regulation of these receptors has been shown, and in particular regulation of the maximum binding capacity of TR by either retinol or RA. Now, using hyperthyroidic rats as a model, the effect of RA on binding capacity and on the mRNA levels of TR and RAR was investigated. The results show that the benefit of vitamin A treatment for the hyperthyroidic state, which has been described for a long time, could be the result of a down-heteroregulation of TR by RA, the active metabolite of retinol.     

Detection of refractive photokeratectomy traces during eye banking: impossible with organ culture but possible with an active storage machine: case report

Cell and Tissue Banking - The detection of corneas operated on for refractive surgery [LASIK or photorefractive keratectomy (PRK)] will become a major concern for eye banks in the coming years...     

A short peptide at the amino terminus of the Sendai virus C protein acts as an independent element that induces STAT1 instability

The Sendai virus C protein acts to dismantle the interferon-induced cellular antiviral state in an MG132-sensitive manner, in part by inducing STAT1 instability. This activity of C maps to the first 23 amino acids (C(1-23)) of the 204-amino-acid (aa)-long protein (C(1-204)). C(1-23) was found to act as an independent viral element that induces STAT1 instability, since this peptide fused to green fluorescent protein (C(1-23)/GFP) is at least as active as C(1-204) in this respect. This peptide also induces the degradation of C(1-23)/GFP and other proteins to which it is fused. Most of C(1-204), and particularly its amino-terminal half, is predicted to be structurally disordered. C(1-23) as a peptide was found to be disordered by circular dichroism, and the first 11 aa have a strong potential to form an amphipathic alpha-helix in low concentrations of trifluoroethanol, which is thought to mimic protein-protein interaction. The critical degradation-determining sequence of C(1-23) was mapped by mutation to eight residues near its N terminus: (4)FLKKILKL(11). All the large hydrophobic residues of (4)FLKKILKL(11), plus its ability to form an amphipathic alpha-helix, were found to be critical for STAT1 degradation. In contrast, C(1-23)/GFP self-degradation did not require (8)ILKL(11), nor the ability to form an alpha-helix throughout this region. Remarkably, C(1-23)/GFP also stimulated C(1-204) degradation, and this degradation in trans required the same peptide determinants as for STAT1. Our results suggest that C(1-204) coordinates its dual activities of regulating viral RNA synthesis and counteracting the host innate antiviral response by sensing both its own intracellular concentration and that of STAT1.     

The amino-terminal extensions of the longer Sendai virus C proteins modulate pY701-Stat1 and bulk Stat1 levels independently of interferon signaling

The Sendai virus (SeV) C proteins are known to interact with Stat1 to prevent interferon (IFN)-induced pY701-Stat1 formation and IFN signaling. Nevertheless, pY701-Stat1 levels paradoxically increase during SeV infection. The C proteins also induce bulk Stat1 instability in some cells, similar to rubulavirus V proteins. We have found that SeV infection increases pY701-Stat1 levels even in cells in which bulk Stat1 levels strongly decrease. Remarkably, both the decrease in bulk Stat1 levels and the increase in pY701-Stat1 levels were found to be independent of the IFN signaling system, i.e., these events occur in mutant cells in which various components of the IFN signaling system have been disabled. Consistent with this, the C-induced decrease in Stat1 levels does not require Y701 of Stat1. We present evidence that C interacts with Stat1 in two different ways, one that prevents IFN-induced pY701-Stat1 formation and IFN signaling that has already been documented, and another that induces pY701-Stat1 formation (while decreasing bulk Stat1 levels) in a manner that does not require IFN signaling. These two types of Stat1 interaction are also distinguishable by C gene mutations. In particular, the IFN signaling-independent Stat1 interactions specifically require the amino-terminal extensions of the longer C proteins. The actions of the SeV C proteins in counteracting the cellular antiviral response are clearly more extensive than previously appreciated.     

C-terminal tail residue Arg1400 enables NADPH to regulate electron transfer in neuronal nitric-oxide synthase

The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg(1400) in the C-terminal tail regulatory element and the 2'-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg(1400) in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent K(m) for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg(1400)-2'-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS.