Direct Vasocontractile Activities of Bupivacaine Enantiomers on the Isolated Rat Thoracic Aorta

Background. In vitro studies with isolated arteries have shown direct vasoactivity of racemic bupivacaine. However, there is little information on the direct vasoactivities of bupivacaine enantiomers, S(−)- and R(+)-bupivacaine. Methods. We performed functional examinations using isolated intact thoracic aortic rings from male Wistar rats. Changes in ring tension produced by S(−)-, R(+)-, or racemic bupivacaine were measured in Krebs solution. Results. S(−)-bupivacaine produced the strongest contraction of the three agents. R(+)-bupivacaine showed limited vasoconstriction. The effects of racemic bupivacaine were located between these two. Conclusion. Each bupivacaine enantiomer showed specific vasocontractile activity, which affects the activity of racemic bupivacaine

Rpd3/CoRest-mediated activity-dependent transcription regulates the flexibility in memory updating in Drosophila

Consolidated memory can be preserved or updated depending on the environmental change. Although such conflicting regulation may happen during memory updating, the flexibility of memory updating may have already been determined in the initial memory consolidation process. Here, we explored the gating mechanism for activity-dependent transcription in memory consolidation, which is unexpectedly linked to the later memory updating in Drosophila. Through proteomic analysis, we discovered that the compositional change in the transcriptional repressor, which contains the histone deacetylase Rpd3 and CoRest, acts as the gating mechanism that opens and closes the time window for activity-dependent transcription. Opening the gate through the compositional change in Rpd3/CoRest is required for memory consolidation, but closing the gate through Rpd3/CoRest is significant to limit future memory updating. Our data indicate that the flexibility of memory updating is determined through the initial activity-dependent transcription, providing a mechanism involved in defining memory state

Direct Vasocontractile Activities of Bupivacaine Enantiomers on the Isolated Rat Thoracic Aorta

Background. In vitro studies with isolated arteries have shown direct vasoactivity of racemic bupivacaine. However, there is little information on the direct vasoactivities of bupivacaine enantiomers, S(−)-and R(+)-bupivacaine. Methods. We performed functional examinations using isolated intact thoracic aortic rings from male Wistar rats. Changes in ring tension produced by S(−)-, R(+)-, or racemic bupivacaine were measured in Krebs solution. Results. S(−)-bupivacaine produced the strongest contraction of the three agents. R(+)-bupivacaine showed limited vasoconstriction. The effects of racemic bupivacaine were located between these two. Conclusion. Each bupivacaine enantiomer showed specific vasocontractile activity, which affects the activity of racemic bupivacaine

Prevention of adverse events of interferon γ gene therapy by gene delivery of interferon γ-heparin-binding domain fusion protein in mice

Sustained gene delivery of interferon (IFN) γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight. These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer. To achieve this, we selected the heparin-binding domain (HBD) of extracellular superoxide dismutase as a molecule to anchor IFNγ to the cell surface. We designed three IFNγ derivatives, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, each of which had 1, 2, or 3 HBDs, respectively. Each plasmid-encoding fusion proteins was delivered to the liver, a model target in this study, by hydrodynamic tail vein injection. The serum concentration of IFNγ-HBD2 and IFNγ-HBD3 after gene delivery was lower than that of IFNγ or IFNγ-HBD1. Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2. Gene delivery of IFNγ-HBD2-suppressed tumor growth in the liver as efficiently as that of IFNγ with much less symptoms of adverse effects. These results indicate that the adverse events of IFNγ gene transfer can be prevented by gene delivery of IFNγ-HBD2, a fusion protein with high cell surface affinity

Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies

Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). Here we characterize global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Bx. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and we identify different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, we find that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. Our results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs

Amounts of lipid mediators produced from WT and iPLA₂γ-KO mouse platelets after ADP stimulation.

<p>Levels of (A) AA, (B) TXB₂ (a TXA₂ metabolite) and 12<i>(S)</i>-HETE, and (C) PGE₂, PGD₂, 6-keto prostaglandin F_1α (6-ketoPGF_1α) (a prostacyclin metabolite) and prostaglandine F_2α (PGF_2α) in supernatants from WT (open columns) or iPLA₂γ-KO (closed columns) platelets stimulated with ADP (10 µM). (D) Levels of TXB₂ in supernatants from WT (open columns) or iPLA₂γ-KO (closed columns) mouse platelets stimulated with collagen (1 µg/ml), thrombin (0.1 U/ml), A23187 (5 µM), PMA (10 nM), AA (100 µM) or U46619 (5 µM). Results are given as mean±SEM (n = 3–9). *<i>P</i><0.05 between iPLA₂γ-KO and WT.</p

Hematological parameters of WT and iPLA₂γ-KO mice.

<p>Data are mean±SEM. No abnormalities or significant differences between WT and iPLA₂γ-KO mice were observed for hematologic parameters (n = 4–5).</p><p>MPV indicates mean platelet volume.</p><p>Hematological parameters of WT and iPLA₂γ-KO mice.</p

iPLA₂γ deficiency inhibits platelet aggregation in response to ADP stimulation.

<p>(A) Representative results from aggregometry testing. Washed platelets from WT (blue) or iPLA₂γ-KO (red) mice were stimulated with the indicated agonists (ADP (10 µM), collagen (1 µg/ml), thrombin (0.1 U/ml), A23187 (5 µM), PMA (10 nM), AA (100 µM) or U46619 (5 µM)), and then light transmission was recorded on an aggregometer. (B) Bar graphs indicate results obtained by aggregometry tests. Washed platelets (WP) or platelets in PRP (200×10³/µl) from WT or iPLA₂γ-KO mice were stimulated with ADP (10 µM), and then light transmission was recorded on an aggregometer. Results are given as the mean percentage of maximum aggregation±SEM (n = 6–10). *<i>P</i><0.05 and **<i>P</i><0.01 between iPLA₂γ-KO and WT.</p

ESI/MS analysis of diacyl-type PE and PC species in WT and iPLA₂γ-KO mouse platelets.

<p>Total lipids were extracted from resting or ADP (10 µM)-stimulated platelet lysates and then subjected to ESI/MS analysis of diacyl-type PE and PC. Levels of (A) diacyl-type PE and (B) PC in resting state of WT (–) or iPLA₂γ-KO platelets (–), or ADP-stimulated WT (ADP) or iPLA₂γ-KO platelets (ADP), and collagen (1 µg/ml)-stimulated WT (collagen) or iPLA₂γ-KO platelets (collagen) (n = 7). Results are given as mean±SEM.</p

P2Y receptors-mediated signaling of iPLA₂γ deficient platelets.

<p>(A and B) Dense granule secretion was evaluated by measuring ATP (A) and serotonin (B) release after the ADP aggregation of WT (open columns) or iPLA₂γ-KO (closed columns) platelets. Results are expressed as the amounts of ATP (n = 10–13) and serotonin (n = 3–4) released by 3.6×10⁷ from platelets. (C) Measuring intraplatelet ATP levels of resting state of WT (open columns) or iPLA₂γ-KO (closed columns) platelets (n = 5). (D) Protein expression of P2Y₁, P2Y₁₂, G_αi, AC and PDEs of platelets from WT and iPLA₂γ-KO mice. β-actin was used as a loading control. Representative results of at least three experiments are shown. (E and F) PRP from WT (blue) or iPLA₂γ-KO (red) mice were stimulated with P2Y₁ agonist (MRS2365) (10 µM) (E) or P2Y₁ antagonist (MRS2279) (10 µM) plus ADP (10 µM) (F), and then light transmission was recorded on an aggregometer. Left panel indicates representative results from aggregometry testing. Right graphs indicate results obtained by aggregometry tests. Results are given as the mean percentage of maximum aggregation±SEM (n = 6–10). *<i>P</i><0.05 between iPLA₂γ-KO and WT. (G) Ca²⁺ influx of WT (open columns) or iPLA₂γ-KO (closed columns) platelets in response to ADP (10 µM). Results are given as mean±SEM (n = 8). (H) PRP (200×10³/µl) from WT (open columns) or iPLA₂γ-KO (closed columns) was incubated both with and without forskolin (FK; 5 µM) for 30 s before ADP (10 µM) was added and the samples were incubated for 5 min at room temperature. Results are given as the mean±SEM (n = 5). *<i>P</i><0.05 between iPLA₂γ-KO and WT.</p