Association of newly synthesized poly(A) polymerase with four distinct polypeptides

Nuclear poly(A) polymerase was isolated from [35S]methionine-labeled hepatoma McA-RH 7777 cells and subjected to DEAE-Sephadex chromatography. Flow-through and low salt wash fractions containing poly(A) polymerase activity were pooled and subjected to immunoblot analysis using anti-tumor type poly(A) polymerase antibodies and a biotinylated second antibody. The immune complex contained a single 48-kDa polypeptide band corresponding to the tumor-type enzyme. When immunoprecipitations were carried out using the same fraction and antibodies, at least five 35S-methionine-labeled proteins with approximate molecular masses of 74, 48, 35, 30, and 22 kDa were observed. Pulse-chase studies did not indicate a precursor-product relationship between the immunoprecipitated proteins. Preimmune sera did not react with poly(A) polymerase or other components in the protein complex. These data show that poly(A) polymerase exists as part of a complex with at least four other polypeptides and suggest that these polypeptides may be involved in the cleavage and/or polyadenylation reactions.     

Na+ channel Scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).     

NMDA Receptor Activation Produces Concurrent Generation of Nitric Oxide and Reactive Oxygen Species: Implications for Cell Death

Abstract: The ability of glutamate to stimulate generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant-sensitive fluorescent dye 2,7-dichlorofluorescin (DCF). Exposure of cells to glutamate (10 µM) produced a rapid generation of oxidants that was blocked ～70% by MK-801 (a noncompetitive NMDA-receptor antagonist). To determine if nitric oxide (NO) or reactive oxygen species (ROS) contributed to the oxidation of DCF, cells were treated with compounds that altered their generation. NO production was inhibited with N^G-nitro-l -arginine methyl ester (l -NAME) (nitric oxide synthase inhibitor) and reduced hemoglobin (NO scavenger). Alternatively, cells were incubated with superoxide dismutase (SOD) and catalase, which selectively metabolize O₂^?· andH₂O₂. Concurrent inhibition of O₂^?· and NO production nearly abolished intracellular oxidant generation. Pretreatment of cells with either chelerythrine (1 µM, protein kinase C inhibitor) or quinacrine (5 µM, phospholipase A₂ inhibitor) before addition of glutamate also blocked oxidation of DCF. Generation of oxidants by glutamate was significantly reduced by incubating the cells in Ca²⁺-free buffer. In cytotoxicity studies, a positive correlation was observed between glutamate-induced death and oxidant generation. Glutamate-induced cytotoxicity was blocked by MK-801 and attenuated by treatment with l -NAME, chelerythrine, SOD, or quinacrine. It is concluded that glutamate induces concurrent generation of NO and ROS by activation of both NMDA receptors and non-NMDA receptors through a Ca²⁺-mediated process. Activation of NO synthase and phospholipaseA₂ contribute significantly to this response. It is proposed that simultaneous generation of NO and ROS results in formation of peroxynitrite, which initiates the cellular damage.     

Modelling evolution on design-by-contract predicts an origin of Life through an abiotic double-stranded RNA world

Background  

It is generally believed that life first evolved from single-stranded RNA (ssRNA) that both stored genetic information and catalyzed the reactions required for self-replication.     

Inhibition of cytomegalovirus-stimulated human cell ornithine decarboxylase by alpha-difluoromethylornithine.

1. The relationship between synthesis of putrescine, human cytomegalovirus DNA synthesis, cell DNA synthesis, and human cytomegalovirus replication has been studied. 2. Stimulation of ornithine decarboxylase activity by shifting low serum-arrested whole human embryo cells to high serum medium is inhibited more than 99% by 2.5 mM DL-alpha-difluoromethylornithine. The addition of DL-alpha-difluoromethylornithine to human cells arrested in low serum and subsequently stimulated by the addition of fresh high serum-containing medium, causes a greater percent inhibition of ornithine decarboxylase activity than when the drug is added to growing human cells. 3. Increased ornithine decarboxylase activity produced by infection of low serum-arrested human cells was inhibited by 5.0 mM of DL-alpha-difluoromethylornithine. However, at a concentration of 5.0 mM, neither DL-alpha-methylornithine nor DL-alpha-difluoromethylornithine affected human cytomegalovirus growth or was toxic to these cells. These data suggest that the increased putrescine synthesis produced by infection is not required for virus replication. 4. The addition of 5.0 mM DL-alpha-difluoromethylornithine had no effect on human cytomegalovirus DNA synthesis or human cytomegalovirus-induced stimulation of cell DNA synthesis. However, 5.0 mM DL-alpha-difluoromethylornithine significantly reduced the stimulation of cell DNA synthesis caused by treatment with mock infecting fluid.     

Elevated hepatic iron: A confounding factor in chronic hepatitis C

Historically, iron overload in the liver has been associated with the genetic disorders hereditary hemochromatosis and thalassemia and with unusual dietary habits. More recently, elevated hepatic iron levels also have been observed in chronic hepatitis C virus (HCV) infection. Iron overload in the liver causes many changes including induction of oxidative stress, damage to lysosomes and mitochondria, altered oxidant defense systems and stimulation of hepatocyte proliferation. Chronic HCV infection causes numerous pathogenic changes in the liver including induction of endoplasmic reticulum stress, the unfolded protein response, oxidative stress, mitochondrial dysfunction and altered growth control. Understanding the molecular and cellular changes that could occur in a liver which has elevated hepatic iron levels and in which HCV replication and gene expression are ongoing has clinical relevance and represents an area of research in need of further investigation.     

Local secondary structure content predicts folding rates for simple,two-state proteins

Many single-domain proteins exhibit two-state folding kinetics, with folding rates that span more than six orders of magnitude. A quantity of much recent interest for such proteins is their contact order, the average separation in sequence between contacting residue pairs. Numerous studies have reached the surprising conclusion that contact order is well-correlated with the logarithm of the folding rate for these small, well-characterized molecules. Here, we investigate the physico-chemical basis for this finding by asking whether contact order is actually a composite number that measures the fraction of local secondary structure in the protein; viz. turns, helices, and hairpins. To pursue this question, we calculated the secondary structure content for 24 two-state proteins and obtained coefficients that predict their folding rates. The predicted rates correlate strongly with experimentally determined rates, comparable to the correlation with contact order. Further, these predicted folding rates are correlated strongly with contact order. Our results suggest that the folding rate of two-state proteins is a function of their local secondary structure content, consistent with the hierarchic model of protein folding. Accordingly, it should be possible to utilize secondary structure prediction methods to predict folding rates from sequence alone.     

Rebound of hepatitis B virus replication in HepG2 cells after cessation of antiviral treatment

Treatment of patients with lamivudine (3TC) results in loss of detectable levels of hepatitis B virus (HBV) DNA from serum; however, the relapse rate, with regard to both reappearance of virus in the bloodstream and hepatic inflammation, is high when therapy is terminated. Although the rebound observed in patients has also been seen in animal hepadnavirus models, rebound has not been analyzed in an in vitro cell culture system. In this study, we used the HBV recombinant baculovirus/HepG2 system to measure the time course of antiviral agent-mediated loss of HBV replication as well as the time course and magnitude of HBV production after release from antiviral treatment. Because of the sensitivity of the system, it was possible to measure secreted virions, intracellular replicative intermediates, and nuclear non-protein-bound HBV DNA and separately analyze individual species of DNA, such as single-stranded HBV DNA compared to the double-stranded form and relaxed circular compared to covalently closed circular HBV DNA. We first determined that HBV replication in the HBV recombinant baculovirus/HepG2 system could proceed for at least 35 days, with a 30-day plateau level of replication, making it possible to study antiviral agent-mediated loss of HBV followed by rebound after cessation of drug treatment. All HBV DNA species decreased in a time-dependent fashion following antiviral treatment, but the magnitude of decline differed for each HBV DNA species, with the covalently closed circular form of HBV DNA being the most resistant to drug therapy. When drug treatment ceased, HBV DNA species reappeared with a pattern that recapitulated the initiation of replication, but with a different time course.     

Transient disruption of intercellular junctions enables baculovirus entry into nondividing hepatocytes

Baculovirus infection has extended the capabilities for transfection of exogenous genes into a variety of mammalian cell types. Because rat hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented chemically defined medium are an excellent model system for studying liver function in vitro, we investigated the ability of baculoviruses to infect and deliver exogenous genes to cells in this culture system. Efficient delivery to hepatocytes in short-term culture becomes restricted to peripheral cells, or "edge" cells, as the hepatocytes acquire intercellular junctions and form islands with time in culture. This barrier to baculovirus entry can be overcome, and the percentage of internal cells within the hepatocyte islands that are infected with the baculovirus can be increased more than 100-fold, when cells are subjected to transient calcium depletion before and during infection. These findings suggest that at least in some cell types, such as hepatocytes, baculovirus entry may require contact with the basolateral surface. We conclude from this study that recombinant baculovirus infection following transient depletion of extracellular calcium results in delivery of exogenous genes to at least 75% of hepatocytes in long-term DMSO culture, thereby making it possible for the first time to carry out gain-of-function and loss-of-function studies in this cell system.