A Comparative Analysis of the Molecular Features of MANF and CDNF.

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system. In this study, co-expression of glucose-regulated protein 78 kDa (GRP78), KDEL receptor 1 or mutant Sar1 into HEK293 cells similarly decreased MANF and CDNF secretion with some degree of variation. Next, we investigated whether CDNF affects the secretion of mouse cysteine-rich with EGF-like domains 2 (CRELD2) because mouse wild-type (wt) MANF but not its KDEL-like motif deleted mutant (ΔCMANF) was found to promote the CRELD2 release from the transfected cells. Co-expressing CRELD2 with wt or ΔC CDNF, we found that CDNF and ΔCMANF hardly elevated the CRELD2 secretion. We then investigated effects of the four or six C-terminal amino acids of MANF and CDNF on the CRELD2 secretion. As a result, co-transfection of mouse CDNF having the mouse MANF-type C-terminal amino acids (CDNFRTDL and CDNFSARTDL) increased the CRELD2 secretion to a small extent, but mouse CDNF having human CDNF-type ones (CDNFKTEL and CDNFHPKTEL) well increased the CRELD2 secretion. On the other hand, the replacement of C-terminal motifs of mouse MANF with those of mouse CDNF (MANFQTEL and MANFYPQTEL) enhanced the CRELD2 secretion, and the mouse MANF having human CDNF-type ones (MANFKTEL and MANFHPKTEL) dramatically potentiated the CRELD2 secretion. These results indicate that the secretion of mouse MANF and mouse CDNF is fundamentally regulated in the same manner and that the variation of four C-terminal amino acids in the MANF and CDNF among species might influence their intracellular functions. This finding could be a hint to identify physiological functions of MANF and CDNF

Peripheral nerve block combined with general anesthesia for lower extremity amputation in hemodialysis patients: case series

Abstract Background Anesthetic management of lower extremity amputation in chronic hemodialysis (HD) patients can be challenging because of their poor cardiovascular status. As previously reported, peripheral nerve block (PNB) may be beneficial in these complicated cases. We report the effects of PNB combined with general anesthesia on hemodynamic stability in HD patients undergoing elective lower extremity amputation. Methods We retrospectively analyzed 13 HD patients who underwent lower extremity amputation. Patients received general anesthesia (GA group, n = 7) or general anesthesia combined with PNB (GA with PNB group, n = 6), as decided by the anesthesiologists. Mean blood pressure (MBP), systolic blood pressure (SBP), lowest BP, heart rate (HR), blood loss, fluid and blood infusion volumes, and doses of vasopressors required were compared for hemodynamic assessment. The coefficient of variation ( CV=σ/X¯ \mathrm{CV}=\upsigma /\overline{\mathcal{X}} ) of MBP (CVMBP) and SBP (CVSBP) was calculated to compare hemodynamic stability. Intraoperative opioid use and postoperative pain scores at rest using a numerical rating scale (NRS) on postoperative days 0 and 1 were compared for pain assessment. We also assessed 30-day mortality. Results CVMBP in the GA group was significantly higher than that in the GA with PNB group (0.15 ± 0.05 and 0.08 ± 0.04, respectively, p = 0.03). The CVSBP in the GA group was also significantly higher than that in the GA with PNB group (0.16 ± 0.02 and 0.09 ± 0.01, respectively, p = 0.03). No significant differences in other hemodynamic parameters were observed. Intraoperative fentanyl doses were significantly lower in the GA with PNB group (GA 210.7 ± 99.9 μg vs. GA with PNB 113.0 ± 75.6 μg, p = 0.04). There were no significant differences in other pain parameters and 30-day mortality between the groups. Conclusion Our results suggest that PNB combined with general anesthesia contributes to intraoperative hemodynamic stability through better pain control in HD patients undergoing lower extremity amputation

Expression of neutral endopeptidase activity during clinical and experimental acute lung injury

Abstract Background Neutral endopeptidase (NEP), an enzyme that cleaves inflammatory bioactive peptides, may play a protective role in the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, its low extracellular activity hinders the precise measurement of changes that take place during ALI/ARDS. The main objective of this study was to clarify the regulation of NEP activity and its expression during ALI/ARDS. Methods In a clinical study, we measured plasma NEP activity in patients who developed postoperative ALI/ARDS, using a HPLC fluorometric system. In an experimental study, we induced ALI by intratracheal instillation of hydrochloric acid (HCl) or lipopolysaccharide (LPS) in mice, and similarly measured NEP activity in plasma, lung tissue, and broncho-alveolar lavage fluid (BALF). We also studied the distribution and measured the amounts of NEP protein, using immuno-histochemical and immunoblot analyses, and measured the levels of NEP mRNA, using real-time reverse transcription-polymerase chain reaction, in the lungs of mice with ALI. Results The plasma NEP activity was significantly lower in patients presenting with ALI/ARDS than in controls. Similarly, the NEP activity in plasma and lung tissue was markedly lower, and lung injuries more severe in LPS- than in HCl-treated mice. In contrast, the activity of NEP in the BALF of LPS-treated mice was increased. The intratracheal instillation of LPS decreased the gene expression of NEP in the lung. Immuno-histochemical and Western immunoblot studies in mice confirmed a) the presence of NEP in the alveolar wall, a critical target in ALI/ARDS, and b) a decrease in its expression in HCl- and LPS-induced ALI. Conclusion In this experimental and clinical study of ALI/ARDS, the activity of NEP was significantly decreased in plasma and increased in the alveolar air space.</p

Reduction of the rocuronium-induced withdrawal reflex by MR13A10A, a generic rocuronium with a novel solution: A randomized, controlled study.

BackgroundRocuronium induces venous pain and the withdrawal reflex during injection. MR13A10A, generic rocuronium with a novel solution, reduced the injection-induced withdrawal reflex in rodents. We hypothesized that MR13A10A would reduce the frequency and severity of injection-induced withdrawal reflexes compared with original rocuronium during clinical anesthesia induction.MethodsThis prospective, open (but assessor-blinded), randomized, controlled study was conducted at a single academic hospital. The assessor was blinded to the study condition in order to minimize observer bias. Participants were allocated to either MR13A10A or traditional formula groups by a blocked stratified randomization method. Participants in the MR13A10A group received MR13A10A, whereas the original rocuronium group received the same amount of original rocuronium. The primary outcome was presence of the withdrawal reflex after rocuronium injection. Severity of the withdrawal reflex, changes in blood pressure and heart rate, and the train of four (TOF) ratio were measured as secondary outcomes. The withdrawal reflex was assessed using a video recording in a blinded manner.ResultsOf the 149 participants, 76 were allocated to the MR13A10A group and 73 to the original rocuronium group. The frequency of the withdrawal reflex was significantly lower with MR13A10A compared with original rocuronium (19.7% and 54.8% for MR13A10A and original rocuronium groups, respectively, pConclusionThe frequency and severity of the withdrawal reflex after injection were significantly reduced by using MR13A10A. MR13A10A might contribute to safe and less invasive anesthesia management

Milnacipran inhibits glutamatergic <it>N</it>-Methyl-D-Aspartate receptor activity in Spinal Dorsal Horn Neurons

Abstract Background Antidepressants, which are widely used for treatment of chronic pain, are thought to have antinociceptive effects by blockade of serotonin and noradrenaline reuptake. However, these drugs also interact with various receptors such as excitatory glutamatergic receptors. Thermal hyperalgesia was induced by intrathecal injection of NMDA in rats. Paw withdrawal latency was measured after intrathecal injection of antidepressants. The effects of antidepressants on the NMDA and AMPA-induced responses were examined in lamina II neurons of rat spinal cord slices using the whole-cell patch-clamp technique. The effects of milnacipran followed by application of NMDA on pERK activation were also investigated in the spinal cord. Results Intrathecal injection of milnacipran (0.1 μmol), but not citalopram (0.1 μmol) and desipramine (0.1 μmol), followed by intrathecal injection of NMDA (1 μg) suppressed thermal hyperalgesia. Milnacipran (100 μM) reduced the amplitude of NMDA (56 ± 3 %, 64 ± 5 % of control)-, but not AMPA (98 ± 5 %, 97 ± 5 % of control)-mediated currents induced by exogenous application and dorsal root stimulation, respectively. Citalopram (100 μM) and desipramine (30 μM) had no effect on the amplitude of exogenous NMDA-induced currents. The number of pERK-positive neurons in the group treated with milnacipran (100 μM), but not citalopram (100 μM) or desipramine (30 μM), followed by NMDA (100 μM) was significantly lower compared with the NMDA-alone group. Conclusions The antinociceptive effect of milnacipran may be dependent on the drug’s direct modulation of NMDA receptors in the superficial dorsal horn. Furthermore, in addition to inhibiting the reuptake of monoamines, glutamate NMDA receptors are also important for analgesia induced by milnacipran.</p