A257T linker region mutant of T7 helicase-primase protein is defective in DNA loading and rescued by T7 DNA polymerase

The helicase and primase activities of the hexameric ring-shaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ～75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.     

Macrophage differentiation and polarization via phosphatidylinositol 3-kinase/Akt-ERK signaling pathway conferred by serum amyloid P component

Macrophage differentiation and polarization is influenced by, and act on, many processes associated with autoimmunity. However, the molecular mechanisms underlying macrophage polarization in systemic lupus erythematosus (SLE) remain largely debated. We previously demonstrated that macrophage M2b polarization conferred by activated lymphocyte-derived (ALD)-DNA immunization could initiate and propagate murine lupus nephritis. Serum amyloid P component (SAP), a conserved acute-phase protein in mice, has been reported to bind to DNA and modulate immune responses. In this study, murine SAP was shown to promote macrophage-mediated ALD-DNA uptake through binding to ALD-DNA (SAP/ALD-DNA). Moreover, macrophage phenotypic switch from a proinflammatory M2b phenotype induced by ALD-DNA alone to an anti-inflammatory M2a phenotype stimulated with SAP/ALD-DNA were found because of PI3K/Akt-ERK signaling activation. Both in vivo SAP supplements and adoptive transfer of ex vivo programmed M2a macrophages induced by SAP/ALD-DNA into SLE mice could efficiently alleviate lupus nephritis. Importantly, increased IL-10 secretion, accompanied by anti-inflammatory effect exerted by M2a macrophages, was found to predominantly impede macrophage M2b polarization. Furthermore, neutralization of IL-10 notably reduced the suppressive effect of M2a macrophages. Our results demonstrate that binding of SAP to ALD-DNA could switch macrophage phenotypic polarization from proinflammatory M2b to anti-inflammatory M2a via PI3K/Akt-ERK signaling activation, thus exerting protective and therapeutic interventions on murine lupus nephritis. These data provide a possible molecular mechanism responsible for modulation of macrophage polarization in the context of lupus nephritis and open a new potential therapeutic avenue for SLE.     

Phenological dynamics of Dasineura mali (Diptera: Cecidomyiidae) and its parasitoid Platygaster demades (Hymenoptera: Platygasteridae) in apple orchards

The midge Dasineura mali Kieffer (Diptera: Cecidomyiidae) is an important pest of apple (Malus domestica Borkh.) and a potential fresh fruit contaminant, causing quarantine concerns. The phenological dynamics of D. mali and its egg parasitoid Platygaster demades Walker (Hymenoptera: Platygasteridae) were studied in the field in Palmerston North, New Zealand, for 2 yr. Both shoot infestation rate by D. mali and D. mali density per shoot sharply increased in the second generation, reaching approximately 65% and 100-200 eggs, respectively. However, although the infestation rate in the third generation remained as high as in the second generation, the pest density per shoot significantly decreased to 40-60 eggs in the third generation. In the fourth generation, both infestation rate and pest density per shoot decreased to approximately 30% and 10 eggs. Due to the simultaneous decline of the apple shoot number and D. mali density in the third and fourth D. mali generations, the absolute number of D. mali in the orchard also has declined proportionally during the same period. The parasitism and superparasitism rates significantly increased as the season progressed, from 45 to 55 and 37% in the first generation to 87 and 82% in the fourth generation, respectively. Our results suggest that P. demades contributes to the continuous decline of D. mali numbers in the field; it is a good searcher, particularly when its hosts become increasingly scarcer over the season, and it avoids overshooting the host population later in the season by increasing superparasitism. The frequency of P. demades aestivation increases from late spring to midsummer and then decreases during the late summer and early autumn. Although the emergence of P. demades was approximately 2 to 3 wk behind that of D. mali in each generation, the increasing parasitism rates from the first to the fourth generations indicate that P. demades is synchronized with D. mali in the field.     

Expression of human equilibrative nucleoside transporter 1 in mouse neurons regulates adenosine levels in physiological and hypoxic-ischemic conditions

Activation of adenosine A(1) receptors inhibits excitatory synaptic transmission. Equilibrative nucleoside transporters (ENTs) regulate extracellular adenosine levels; however, the role of neuronal ENTs in adenosine influx and efflux during cerebral ischemia has not been determined. We used mice with neuronal expression of human ENT type 1 and wild type (Wt) littermates to compare responses to in vitro hypoxic or ischemic conditions. Extracellular recordings in the CA1 region of hippocampal slices from transgenic (Tg) mice revealed increased basal synaptic transmission, relative to Wt slices, and an absence of 8-cyclopentyl-1,3-dipropyl-xanthine mediated augmentation of excitatory neurotransmission. Adenosine (10-100 μM) had a reduced potency for inhibiting synaptic transmission in slices from Tg mice; inhibitory concentration 50% values were approximately 25 and 50 μM in Wt and Tg slices, respectively. Potency of the A(1) receptor agonist N(6) -cyclopentyladenosine (1 nM-1 μM) was unchanged. Transient hypoxia or oxygen-glucose deprivation produced greater inhibition of excitatory neurotransmission in slices from Wt than Tg, mice. The ENT1 inhibitor S-(4-nitrobenzyl)-6-thioinosine abolished these differences. Taken together, our data provide evidence that neuronal ENTs reduce hypoxia- and ischemia-induced increases in extracellular adenosine levels and suggest that inhibition of neuronal adenosine transporters may be a target for the treatment of cerebral ischemia.     

Chemokine CCL2 modulation of neuronal excitability and synaptic transmission in rat hippocampal slices

In addition to its well-characterized effects in immune system, chemokine CC motif ligand 2 (CCL2, formerly known as monocyte chemoattractant protein-1) is believed to play an important role in brain physiological and pathological processes. It has been shown that CCL2 and its cognate receptor chemokine CC motif receptor 2 are constitutively expressed in several brain regions including the hippocampus, and the expression is up-regulated under pathological conditions. Whereas most investigations have so far focused on its involvement in CNS pathology, few studies have examined the effects of CCL2 on neuronal and synaptic physiology. In this study, we tested the effects of CCL2 on neuronal excitability and excitatory synaptic transmission in the CA1 region of rat hippocampal slices using whole-cell patch clamp techniques. Bath application of CCL2 depolarized membrane potential and increased spike firing in CA1 neuronal cells. Bath application of CCL2 also produced an increase of excitatory post-synaptic currents recorded in Schaffer-collateral fibers to CA1 synapses. Quantal analysis revealed that CCL2 increased the frequency of spontaneous excitatory post-synaptic current occurrence and mean quantal content. Taken together, our data indicate that CCL2 enhances neuronal excitability and synaptic transmission via pre-synaptic mechanisms. These results support the emerging concept that chemokines function as neuromodulators in the CNS.     

Benzo(a)pyrene-induced mitochondrial dysfunction and cell death in p53-null Hep3B cells

Benzo(a)pyrene (BaP) has been shown to induce apoptosis and necrosis in various cell types. However, the effect of BaP on mitochondria function and p73, and their possible roles in BaP-induced cell death have not been well studied. This study focused on mitochondria-mediated cell death and the occurrence of p73 protein accumulation in BaP-treated human hepatoma Hep3B (p53-null) cells. We found that BaP (8, 16, 32 and 64μM) induced early necrosis at 12h and delayed apoptosis at 24h. BaP dramatically induced ethoxyresorufin-O-deethylase activity and led to significant increase in oxidative stress at early time points (6 and 12h). Necrotic cell death was concurrent with loss of mitochondrial membrane potential, decrease in the ATP level and activities of Na(+)/K(+)-ATPase and Ca(2+)/Mg(2+)-ATPase. However, these changes were reversed in the process of apoptosis. In addition, after BaP treatment, c-Jun N-terminal kinase (JNK) and Bax were activated during apoptosis and no change in p73 protein level was observed. These results revealed that the cells with mitochondria dysfunction and ATP depletion underwent necrosis at early time point and apoptosis afterward when they recovered from mitochondrial dysfunction and ATP depletion. Activation of JNK and Bax possibly contributed to BaP-induced apoptosis.     

Feedback regulation of endothelial cell surface plasmin generation by PKC-dependent phosphorylation of annexin A2

In response to blood vessel injury, hemostasis is initiated by platelet activation, advanced by thrombin generation, and tempered by fibrinolysis. The primary fibrinolytic protease, plasmin, can be activated either on a fibrin-containing thrombus or on cells. Annexin A2 (A2) heterotetramer (A2·p11)(2) is a key profibrinolytic complex that assembles plasminogen and tissue plasminogen activator and promotes plasmin generation. We now report that, in endothelial cells, plasmin specifically induces activation of conventional PKC, which phosphorylates serine 11 and serine 25 of A2, triggering dissociation of the (A2·p11)(2) tetramer. The resulting free p11 undergoes ubiquitin-mediated proteasomal degradation, thus preventing further translocation of A2 to the cell surface. In vivo, pretreatment of A2(+/+) but not A2(-/-) mice with a conventional PKC inhibitor significantly reduced thrombosis in a carotid artery injury model. These results indicate that augmentation of fibrinolytic vascular surveillance by blockade of serine phosphorylation is A2-dependent. We also demonstrate that plasmin-induced phosphorylation of A2 requires both cleavage of A2 and activation of Toll-like receptor 4 on the cell surface. We propose that plasmin can limit its own generation by triggering a finely tuned "feedback" mechanism whereby A2 becomes serine-phosphorylated, dissociates from p11, and fails to translocate to the cell surface.     

Overexpression of the Arabidopsis ��-expansin gene AtEXPA1 accelerates stomatal opening by decreasing the volumetric elastic modulus

Guard cell walls of stomata are highly specialized in plants. Previous research focused on the structure and anatomy of guard cell walls, but little is known about guard cell regulation during stomata movement. In this work, we investigate the possible biological role of the Arabidopsis expansin gene AtEXPA1 in stomatal opening. The AtEXPA1 promoter drove the expression of the GUS reporter gene specifically in guard cells. Light-induced stomatal opening was accelerated in 35S::AtEXPA1 lines, whereas the anti-AtEXPA1 antibody decelerated light-induced stomatal opening. The inhibition of the anti-AtEXPA1 antibody on stomatal opening was largely dependent on the environmental pH. The volumetric elastic modulus (ε) was measured as an indicator of changes in the cell wall. The ε value of guard cells in 35S::AtEXPA1 lines was smaller than in the wild types. The putative role of AtEXPA1 as controller of stomatal opening rate and its regulation are discussed.     

Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue

New platforms allow quantification of gene expression from large, replicated experiments but current sampling protocols for plant tissue using immediate flash freezing in liquid nitrogen are a barrier to these high-throughput studies. In this study, we compared four sampling methods for RNA extraction for gene expression analysis: (1) the standard sampling method of flash freezing whole leaves in liquid nitrogen immediately upon removal from the plant; (2) incubation of excised leaf disks for 2 min at field temperature followed by flash freezing; (3) incubation of excised leaf disks for 1 h on ice followed by flash freezing; and (4) incubation of excised leaf disks for 1 h at field temperature followed by flash freezing. Gene expression analysis was done for 23 genes using nCounter, and normalization of the data was done using the geometric mean of five housekeeping genes. Quality of RNA was highest for protocol A and lowest for protocol D. Despite some differences in RNA quality, gene expression was not significantly different among protocols A, B, and C for any of the 23 genes. Expression of some genes was significantly different between protocol D and the other protocols. This study demonstrates that when sampling leaf disks for gene expression analysis, the time between tissue removal from the plant and flash freezing in liquid nitrogen can be extended. This increase in time allowable during sampling provides greater flexibility in sampling large replicated field experiments for statistical analysis of gene expression data.