Poly(vinyl amine)-silica composite nanoparticles: models of the silicic acid cytoplasmic pool and as a silica precursor for composite materials formation

The role of polymer (poly(vinylamine)) size (238-11000 units) on silicic acid condensation to yield soluble nanoparticles or composite precipitates has been explored by a combination of light scattering (static and dynamic), laser ablation combined with aerosol spectrometry, IR spectroscopy, and electron microscopy. Soluble nanoparticles or composite precipitates are formed according to the degree of polymerization of the organic polymer and pH. Nanoparticles prepared in the presence of the highest molecular weight polymers have core-shell like structures with dense silica cores. Composite particles formed in the presence of polymers with extent of polymerization below 1000 consist of associates of several polymer-silica nanoparticles. The mechanism of stabilization of the "soluble" silica particles in the tens of nanometer size range involves cooperative interactions with the polymer chains which varies according to chain length and pH. An example of the use of such polymer-poly(silicic acid) nanoparticles in the generation of composite polymeric materials is presented. The results obtained have relevance to the biomimetic design of new composite materials based on silica and polymers and to increasing our understanding of how silica may be manipulated (stored) in the biological environment prior to the formation of stable mineralized structures. We suspect that a similar method of storing silicic acid in an active state is used in silicifying organisms, at least in diatom algae.     

Nse1 RING-like domain supports functions of the Smc5-Smc6 holocomplex in genome stability

The Smc5-Smc6 holocomplex plays essential but largely enigmatic roles in chromosome segregation, and facilitates DNA repair. The Smc5-Smc6 complex contains six conserved non-SMC subunits. One of these, Nse1, contains a RING-like motif that often confers ubiquitin E3 ligase activity. We have functionally characterized the Nse1 RING-like motif, to determine its contribution to the chromosome segregation and DNA repair roles of Smc5-Smc6. Strikingly, whereas a full deletion of nse1 is lethal, the Nse1 RING-like motif is not essential for cellular viability. However, Nse1 RING mutant cells are hypersensitive to a broad spectrum of genotoxic stresses, indicating that the Nse1 RING motif promotes DNA repair functions of Smc5-Smc6. We tested the ability of both human and yeast Nse1 to mediate ubiquitin E3 ligase activity in vitro and found no detectable activity associated with full-length Nse1 or the isolated RING domains. Interestingly, however, the Nse1 RING-like domain is required for normal Nse1-Nse3-Nse4 trimer formation in vitro and for damage-induced recruitment of Nse4 and Smc5 to subnuclear foci in vivo. Thus, we propose that the Nse1 RING-like motif is a protein–protein interaction domain required for Smc5-Smc6 holocomplex integrity and recruitment to, or retention at, DNA lesions.     

HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.     

Sequence-tagged-site (STS) markers of arbitrary genes: the utility of black spruce-derived STS primers in other conifers

 Sequence-tagged-site primers, previously developed based upon black spruce (Picea mariana) cDNA sequences, were tested for their ability to direct specific amplification in two individuals of each of 12 additional conifer species. Nearly all (95–97%) of the primers functioned well in congeneric trials, while a lower proportion (21–33%) scored positively in other Pinaceae genera. Outside of the Pinaceae, amplification of homologous products was not achieved. Products from the various species often differed in size from their homologs in black spruce. In one case a large difference in size was due to the lack of an intron in a jack pine product while in several other cases the differences were due to the presence or absence of large direct repeats in the DNA sequences. Length polymorphism was occasionally evident between the two individuals examined of a given species. We investigated marker polymorphism in detail in a panel of 15 white spruce (Picea glauca) trees. Allelic segregation among haploid megagametophytes was revealed directly at 16 loci by standard agarose-gel electrophoresis without any additional manipulation of amplification products. Polymorphisms observed at 12 of these loci were exclusively co-dominant. For this subset of 12 loci, the average number of alleles was 3.2 and the average observed heterozygosity was 0.37. Received: 10 April 1998 / Accepted: 22 April 1998     

Extrahepatic cells contribute to the progenitor/stem cell response following reduced-size liver transplantation in mice

The extent to which extrahepatic cells participate in liver regeneration following transplantation is not known. Either full-size or reduced-size livers from wild-type mice were implanted into green fluorescent protein-positive (GFP(+)) transgenic recipient mice to determine whether regenerated liver contained host-derived GFP(+) hepatic cells. After reduced-size liver transplantation, GFP(+) cells were localized to the portal zone of the liver lobule. Interestingly, GFP(+) cells stained for CD117, a marker for progenitor cells, beginning 2 days after transplantation. A significant number of GFP(+) CD117(+) cells were identified in donor livers after 28 days. GFP(+) cells comprised nearly 9% of the donor liver 28 days after reduced-size liver transplant. Moreover, GFP(+) cells also expressed the hepatic progenitor cell marker A6 and novel marker hepatic-specific antigen (HSA), as well as stem cell antigen-1 (Sca-1). Interestingly, some GFP(-) cells also were stained for CD117 and A6, suggesting that both extrahepatic and intrahepatic stem cells were present and may have contributed to the regenerative response under these conditions. Reduced-size liver transplantation using GFP(+) transgenic mice supports the hypothesis that recipient-derived progenitor cells are present and may contribute to liver regeneration following transplantation.     

Reduced Glomerular Filtration Rate and Its Association with Clinical Outcome in Older Patients at Risk of Vascular Events: Secondary Analysis

Background

Reduced glomerular filtration rate (GFR) is associated with increased cardiovascular risk in young and middle aged individuals. Associations with cardiovascular disease and mortality in older people are less clearly established. We aimed to determine the predictive value of the GFR for mortality and morbidity using data from the 5,804 participants randomized in the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER).

Methods and Findings

Glomerular filtration rate was estimated (eGFR) using the Modification of Diet in Renal Disease equation and was categorized in the ranges ([20–40], [40–50], [50–60]) ≥ 60 ml/min/1.73 m². Baseline risk factors were analysed by category of eGFR, with and without adjustment for other risk factors. The associations between baseline eGFR and morbidity and mortality outcomes, accrued after an average of 3.2 y, were investigated using Cox proportional hazard models adjusting for traditional risk factors. We tested for evidence of an interaction between the benefit of statin treatment and baseline eGFR status. Age, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, C-reactive protein (CRP), body mass index, fasting glucose, female sex, histories of hypertension and vascular disease were associated with eGFR (p = 0.001 or less) after adjustment for other risk factors. Low eGFR was independently associated with risk of all cause mortality, vascular mortality, and other noncancer mortality and with fatal and nonfatal coronary and heart failure events (hazard ratios adjusted for CRP and other risk factors (95% confidence intervals [CIs]) for eGFR < 40 ml/min/1.73m² relative to eGFR ≥ 60 ml/min/1.73m² respectively 2.04 (1.48–2.80), 2.37 (1.53–3.67), 3.52 (1.78–6.96), 1.64 (1.18–2.27), 3.31 (2.03–5.41). There were no nominally statistically significant interactions (p < 0.05) between randomized treatment allocation and eGFR for clinical outcomes, with the exception of the outcome of coronary heart disease death or nonfatal myocardial infarction (p = 0.021), with the interaction suggesting increased benefit of statin treatment in subjects with impaired GFRs.

Conclusions

We have established that, in an elderly population over the age of 70 y, impaired GFR is associated with female sex, with presence of vascular disease, and with levels of other risk factors that would be associated with increased risk of vascular disease. Further, impaired GFR is independently associated with significant levels of increased risk of all cause mortality and fatal vascular events and with composite fatal and nonfatal coronary and heart failure outcomes. Our analyses of the benefits of statin treatment in relation to baseline GFR suggest that there is no reason to exclude elderly patients with impaired renal function from treatment with a statin.     

Discovery of a thermophilic protein complex stabilized by topologically interlinked chains

A growing number of organisms have been discovered inhabiting extreme environments, including temperatures in excess of 100 degrees C. How cellular proteins from such organisms retain their native folds under extreme conditions is still not fully understood. Recent computational and structural studies have identified disulfide bonding as an important mechanism for stabilizing intracellular proteins in certain thermophilic microbes. Here, we present the first proteomic analysis of intracellular disulfide bonding in the hyperthermophilic archaeon Pyrobaculum aerophilum. Our study reveals that the utilization of disulfide bonds extends beyond individual proteins to include many protein-protein complexes. We report the 1.6 A crystal structure of one such complex, a citrate synthase homodimer. The structure contains two intramolecular disulfide bonds, one per subunit, which result in the cyclization of each protein chain in such a way that the two chains are topologically interlinked, rendering them inseparable. This unusual feature emphasizes the variety and sophistication of the molecular mechanisms that can be achieved by evolution.     

Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been used to identify its phosphorylation sites. This article highlights the recent progress and different approaches utilized for the identification of phosphorylation sites in mammalian TTP. Important but limited results are obtained using traditional methods, including in vivo labeling, site-directed mutagenesis, phosphopeptide mapping and protein sequencing. Mass spectrometry (MS), including MALDI/MS, MALDI/MS/MS, liquid chromatography/MS/MS, immobilized metal ion affinity chromatography (IMAC)/MALDI/MS/MS and multidimensional protein identification technology has led the way in identifying TTP phosphorylation sites. The combination of these approaches has identified multiple phosphorylation sites in mammalian TTP, some of which are predicted by motif scanning to be phosphorylated by several protein kinases. This information should provide the molecular basis for future investigation of TTP's regulatory functions in controlling proinflammatory cytokines.