Purification and characterization of organellar DNA polymerases in the red alga Cyanidioschyzon merolae

DNA polymerase gamma, a mitochondrial replication enzyme of yeasts and animals, is not present in photosynthetic eukaryotes. Recently, DNA polymerases with distant homology to bacterial DNA polymerase I were reported in rice, Arabidopsis, and tobacco, and they were localized to both plastids and mitochondria. We call them plant organellar DNA polymerases (POPs). However, POPs have never been purified in the native form from plant tissues. The unicellular thermotrophic red alga Cyanidioschyzon merolae contains two genes encoding proteins related to Escherichia coli DNA polymerase I (PolA and PolB). Phylogenetic analysis revealed that PolB is an ortholog of POPs. Nonphotosynthetic eukaryotes also have POPs, which suggested that POPs have an ancient origin before eukaryotic photosynthesis. PolA is a homolog of bacterial DNA polymerase I and is distinct from POPs. PolB was purified from the C. merolae cells by a series of column chromatography steps. Recombinant protein of PolA was also purified. Sensitivity to inhibitors of DNA synthesis was different in PolA, PolB, and E. coli DNA polymerase I. Immunoblot analysis and targeting studies with green fluorescent protein fusion proteins demonstrated that PolA was localized in the plastids, whereas PolB was present in both plastids and mitochondria. The expression of PolB was regulated by the cell cycle. The available results suggest that PolB is involved in the replication of plastids and mitochondria.     

Autotaxin, a synthetic enzyme of lysophosphatidic acid (LPA), mediates the induction of nerve-injured neuropathic pain

The extracellular signal-regulated kinase (ERK) cascade has been shown to be a key modulator of pain processing in the central nucleus of the amygdala (CeA) in mice. ERK is activated in the CeA during persistent inflammatory pain and this activation is both necessary and sufficient to induce peripheral tactile hypersensitivity. Interestingly, biochemical studies show that inflammation-induced ERK activation in the CeA only occurs in the right, but not the left hemisphere. This inflammation-induced ERK activation in the right CeA is independent of the side of peripheral inflammation, suggesting that there is a dominant role of the right hemisphere in the modulation of pain by ERK activation in the CeA. However, the functional significance of this biochemical lateralization has yet to be determined. In the present study, we tested the hypothesis that modulation of pain by ERK signaling in the CeA is functionally lateralized. We acutely blocked ERK activation in the CeA by infusing the MEK inhibitor U0126 into the right or the left hemisphere and then measured the behavioral effects on inflammation-induced mechanical hypersensitivity in mice. Our results show that blockade of ERK activation in the right, but not the left CeA, decreases inflammation-induced peripheral hypersensitivity independent of the side of peripheral injury. These findings demonstrate that modulation of pain by ERK signaling in the CeA is functionally lateralized to the right hemisphere, suggesting a dominant role of the right amygdala in pain processing.     

Artificial restriction DNA cutter for site-selective scission of double-stranded DNA with tunable scission site and specificity

The artificial restriction DNA cutter (ARCUT) method to cut double-stranded DNA at designated sites has been developed. The strategy at the base of this approach, which does not rely on restriction enzymes, is comprised of two stages: (i) two strands of pseudo-complementary peptide nucleic acid (pcPNA) anneal with DNA to form 'hot spots' for scission, and (ii) the Ce(IV)/EDTA complex acts as catalytic molecular scissors. The scission fragments, obtained by hydrolyzing target phosphodiester linkages, can be connected with foreign DNA using DNA ligase. The location of the scission site and the site-specificity are almost freely tunable, and there is no limitation to the size of DNA substrate. This protocol, which does not include the synthesis of pcPNA strands, takes approximately 10 d to complete. The synthesis and purification of the pcPNA, which are covered by a related protocol by the same authors, takes an additional 7 d, but pcPNA can also be ordered from custom synthesis companies if necessary.     

Correspondence between food consistency and suprahyoid muscle activity, tongue pressure, and bolus transit times during the oropharyngeal phase of swallowing.

This study aimed to evaluate the effects of food texture and viscosity on the swallowing function by measuring tongue pressure and performing a videofluorographic (VF) examination. Eleven normal adults were recruited for this study. Test foods with different consistencies and liquid contents, i.e., a half-solid nutrient made of 0.8 and 1.5% agar powder, syrup, and a liquid containing 40 wt/vol% barium sulfate, were swallowed, and the anterior (AT) and posterior tongue pressures (PT) and electromyographic (EMG) activity of the suprahyoid muscles were recorded, together with VF images. The timing of each event obtained from EMG, tongue pressure, and VF recordings was measured and then compared. We found that the AT and PT activity patterns were similar and showed a single peak. The peak, area, and time duration of all of the variables for AT and PT and EMG burst increased with increasing hardness of the bolus. The onset of the EMG burst always preceded those of the AT and PT activities, while there were no significant differences in peak and offset times among EMG burst, AT, and PT. Total swallowing time and oral ejection time were significantly longer during the swallowing of 1.5% agar than any other boluses, while pharyngeal transit time and clearance time were significantly longer during the swallowing of syrup, which was as hard as the liquid, but showed a higher viscosity than the liquid. The results suggested that the major effects of food hardness were to delay oral ejection time, which strongly delays total swallowing time. In addition, pharyngeal bolus transit is not dependent on the hardness of food but on its viscosity.     

Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.     

Human T-cell leukemia virus type 1 HBZ protein bypasses the targeting function of ubiquitination

Human T-cell leukemia virus type 1 (HTLV-1) encodes an antisense viral gene product termed HTLV-1 basic leucine-zipper factor (HBZ). HBZ forms heterodimers with c-Jun, a member of the AP-1 family, and promotes its proteasomal degradation. Although most proteasomal substrates are targeted for degradation via conjugation of polyubiquitin chains, we show that ubiquitination is not required for HBZ-mediated proteasomal degradation of c-Jun. We demonstrate that HBZ directly interacts with both the 26 S proteasome and c-Jun and facilitates the delivery of c-Jun to the proteasome without ubiquitination. HBZ acts as a tethering factor between the 26 S proteasome and its substrate, thereby bypassing the targeting function of ubiquitination. These findings disclose a novel viral strategy to utilize the cellular proteolytic system for viral propagation.     

Porcine dentin sialophosphoprotein: length polymorphisms, glycosylation, phosphorylation, and stability

Dentin sialophosphoprotein (DSPP) is critical for proper mineralization of tooth dentin, and mutations in DSPP cause inherited dentin defects. Dentin phosphoprotein (DPP) is the C-terminal cleavage product of DSPP that binds collagen and induces intrafibrillar mineralization. We isolated DPP from individual pigs and determined that its N-terminal and C-terminal domains are glycosylated and that DPP averages 155 phosphates per molecule. Porcine DPP is unstable at low pH and high temperatures, and complexing with collagen improves its stability. Surprisingly, we observed DPP size variations on SDS-PAGE for DPP isolated from individual pigs. These variations are not caused by differences in proteolytic processing or degrees of phosphorylation or glycosylation, but rather to allelic variations in Dspp. Characterization of the DPP coding region identified 4 allelic variants. Among the 4 alleles, 27 sequence variations were identified, including 16 length polymorphisms ranging from 3 to 63 nucleotides. None of the length variations shifted the reading frame, and all localized to the highly redundant region of the DPP code. The 4 alleles encode DPP domains having 551, 575, 589, or 594 amino acids and completely explain the DPP size variations. DPP length variations are polymorphic and are not associated with dentin defects.     

Wnt/beta-catenin signaling controls development of the blood-brain barrier

The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.