The effect of G-CSF in a myocardial ischemia reperfusion model rat

Purpose : It has been reported that G-CSF administration improves cardiac function by reducing the area of the infarct in a myocardial infarction model rat. In the present study, myocardial infarction model rats, produced by ligation of the left anterior coronary artery, were prepared. The G-CSF effect for treating cardiac muscle cell disorders by ischemia reperfusion was studied. Methods : Myocardial infarction model rats were produced by ligation of the left anterior descending coronary artery in 12-week-old Wistar rats. G-CSF was administered subcutaneously daily at a dose of 100 μg/kg/day for 5 days to rats with a complete ligation (MI-G group, n=6) and rats in which the ligated coronary artery was reperfused 30 minutes after the ligation (R-G group, n=6). Physiological saline was subcutaneously administered to rats with a complete ligation and reperfusion (MI-C and R-C groups, respectively, n=6 each), as controls. After 4 weeks, the infarct area ratio (%), cardiac function on echocardiography (left ventricular ejection fraction), and a myocardial histopathological diagnosis were carried out and the results compared among the groups. Results : No significant differences were found in the proportion of the residual heart muscle in the infarct lesion, myocardial wall thickness, or left ventricular ejection fraction between the MI-G and MI-C groups. In contrast, the infarct area, myocardial wall thickness, and left ventricular ejection fraction were significantly improved in the R-G group compared to the R-C group (p<0.05). Conclusions : Any inhibitory effect of G-CSF on the infarct lesion was found in the myocardial infarction reperfusion model rat, but only a small effect was found in rats with a complete ligation-induced myocardial infarction. The findings in the present study, therefore, suggest that G-CSF is effective for treating cardiac muscle cell disorders by ischemia reperfusion

In vitro substrate phosphorylation by Ca2+/calmodulin-dependent protein kinase kinase using guanosine-5[prime]-triphosphate as a phosphate donor

BACKGROUND: Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases — including CaMKI, CaMKIV, and AMPK— to stimulate multiple Ca(2+)-signal transduction pathways. To identify previously unidentified CaMKK substrates, we used various nucleotides as phosphate donors to develop and characterize an in vitro phosphorylation assay for CaMKK. RESULTS: Here, we found that the recombinant CaMKK isoforms were capable of utilizing Mg-GTP as a phosphate donor to phosphorylate the Thr residue in the activation-loop of CaMKIα (Thr(177)) and of AMPK (Thr(172)) in vitro. Kinetic analysis indicated that the K(m) values of CaMKK isoforms for GTP (400-500 μM) were significantly higher than those for ATP (~15 μM), and a 2- to 4-fold decrease in V(max) was observed with GTP. We also confirmed that an ATP competitive CaMKK inhibitor, STO-609, also competes with GTP to inhibit the activities of CaMKK isoforms. In addition, to detect enhanced CaMKI phosphorylation in brain extracts with Mg-GTP and recombinant CaMKKs, we found potential CaMKK substrates of ~45 kDa and ~35 kDa whose Ca(2+)/CaM-induced phosphorylation was inhibited by STO-609. CONCLUSIONS: These results indicated that screens that use STO-609 as a CaMKK inhibitor and Mg-GTP as a CaMKK-dependent phosphate donor might be useful to identify previously unidentified downstream target substrates of CaMKK

Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells

To assess the isoform specificity of the Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKKα mutant (Ala292Thr/Leu233Phe) and a CaMKKβ mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2–3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5′-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wild-type or the STO-609-resistant mutant of CaMKKα was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKKβ mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKKβ is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound

New insertion method of transnasal ileus tube for small bowel obstruction: Anterior balloon method.

BACKGROUND:Small bowel obstruction (SBO) is usually caused by postoperative adhesions and malignant disease, and decompression is effective for SBO. Our previous case report suggested that a new transnasal ileus tube insertion method, the anterior balloon method (ABM), could achieve decompression for adhesive SBO. AIMS:The study aimed to investigate the effectiveness of a new method for inserting transnasal ileus tubes in patients with SBO. METHODS:Altogether, 134 patients with small bowel obstruction treated from January 2011 to December 2017 were reviewed. The patients were categorized into two groups: those with the new method that inserts an anterior balloon (ABM group: 52 patients, 2014-2017) versus those with the ordinary insertion method (OIM group: 82 patients, 2011-2014). RESULTS:The patients' characteristics and symptoms on admission were similar in the ABM and OIM groups. Adhesions were the main cause of ileus in the two groups. The insertion time duration was significantly shorter in the ABM group than in OIM group (28.4 ± 9.1 vs. 33.5 ± 13.0 min; p = 0.01). The ABM group also had significantly longer tubes than OIM group (222.4 ± 32.2 vs. 157.4 ± 31.7 cm; p < 0.001), which resulted in a significantly shorter time until clinical symptoms were relieved in ABM group. There were no significant differences in adverse events between the two groups. CONCLUSIONS:The ABM group had shorter insertion duration and longer tubes than those of OIM group. The ABM might become a preferred therapeutic choice to achieve decompression in patients with SBO

Is the Efficacy of Adding Ramucirumab to Docetaxel Related to a History of Immune Checkpoint Inhibitors in the Real-World Clinical Practice?

Reports on the efficacy of second-line treatment with cytotoxic agents after treatment with immune checkpoint inhibitors are limited. Here, we retrospectively evaluated patients in the real-world clinical practice treated with docetaxel or docetaxel plus ramucirumab. Ninety-three patients treated with docetaxel or docetaxel plus ramucirumab as a second- or later-line therapy were included. The patients were categorized into the following four treatment groups: docetaxel group (n = 50), docetaxel/ramucirumab group (n = 43) and pretreated (n = 45) and untreated (n = 48) with immune checkpoint inhibitor groups. The docetaxel/ramucirumab group showed an overall response rate of 57.1% in patients pretreated with immune checkpoint inhibitors and 20% in untreated patients. The docetaxel group showed an overall response rate of 15.4% in patients pretreated with immune checkpoint inhibitors and 5.0% in untreated patients. The median time-to-treatment failure and the median survival time were longer in the docetaxel/ramucirumab group than in the docetaxel group in both immune checkpoint inhibitor-pretreated and -untreated groups. There was no difference in time-to-treatment failure and overall survival between immune checkpoint inhibitor-pretreated and -untreated groups in each docetaxel and docetaxel/ramucirumab treatment group. In conclusion, our real-world data show that the addition of ramucirumab to docetaxel was superior to docetaxel monotherapy for improving time-to-treatment failure and overall survival, irrespective of previous treatment with immune checkpoint inhibitors