Calreticulin inhibits the MEK1,2-ERK1,2 pathway in alpha 1-adrenergic receptor/Gh-stimulated hypertrophy of neonatal rat cardiomyocytes

In cardiac myocytes, stimulation of alpha(1)-adrenoceptor (AR) leads to a hypertrophic phenotype. The G(h) protein (transglutaminase II, TGII) is tissue type transglutaminase and transmits the alpha(1B)-adrenoceptor signal with GTPase activity. Recently, it has been shown that the calreticulin (CRT) down-regulates both GTP binding and transglutaminase activities of TGII. To elucidate whether G(h) mediates norepinephrine-stimulated intracellular signal transductions leading to activation of extracellular signal-regulated kinases (ERKs) and neonatal rat cardiomyocyte hypertrophy, we examined the effects of G(h) on the activation of ERKs and inhibitory effects of CRT on alpha(1)-adrenoceptor/G(h) signaling. In neonatal rat cardiomyocytes, norepinephrine-induced ERKs activation was inhibited by an alpha(1)-adrenoceptor blocker (prazosin), but not by an beta-adrenoceptor blocker (propranolol). Overexpression of the G(h) protein stimulated norepinephrine-induced ERKs activation, which was inhibited by alpha-adrenoceptor blocker (prazosin). Co-overexpression of G(h) and CRT abolished norepinephrine-induced ERKs activation. Taken together, norepinephrine induces hypertrophy in neonatal rat cardiomyocytes through alpha(1)-AR stimulation and G(h) is partly involved in norepinephrine-induced MEK1,2/ERKs activation. Activation of G(h)-mediated MEK1,2/ERKs was completely inhibited by CRT.     

Bioreactance Is Not Interchangeable with Thermodilution for Measuring Cardiac Output during Adult Liver Transplantation

BackgroundThermodilution technique using a pulmonary artery catheter is widely used for the assessment of cardiac output (CO) in patients undergoing liver transplantation. However, the unclearness of the risk-benefit ratio of this method has led to an interest in less invasive modalities. Thus, we evaluated whether noninvasive bioreactance CO monitoring is interchangeable with thermodilution technique.MethodsNineteen recipients undergoing adult-to-adult living donor liver transplantation were enrolled in this prospective observational study. COs were recorded automatically by the two devices and compared simultaneously at 3-minute intervals. The Bland–Altman plot was used to evaluate the agreement between bioreactance and thermodilution. Clinically acceptable agreement was defined as a percentage error of limits of agreement <30%. The four quadrant plot was used to evaluate concordance between bioreactance and thermodilution. Clinically acceptable concordance was defined as a concordance rate >92%.ResultsA total of 2640 datasets were collected. The mean CO difference between the two techniques was 0.9 l/min, and the 95% limits of agreement were -3.5 l/min and 5.4 l/min with a percentage error of 53.9%. The percentage errors in the dissection, anhepatic, and reperfusion phase were 50.6%, 56.1%, and 53.5%, respectively. The concordance rate between the two techniques was 54.8%.ConclusionBioreactance and thermodilution failed to show acceptable interchangeability in terms of both estimating CO and tracking CO changes in patients undergoing liver transplantation. Thus, the use of bioreactance as an alternative CO monitoring to thermodilution, in spite of its noninvasiveness, would be hard to recommend in these surgical patients.     

Mannitol induces selective astroglial death in the CA1 region of the rat hippocampus following status epilepticus

In the present study, we addressed the question of whether treatment with mannitol, an osmotic diuretic, affects astrogliovascular responses to status epilepticus (SE). In saline-treated animals, astrocytes exhibited reactive astrogliosis in the CA1-3 regions 2-4 days after SE. In the mannitol-treated animals, a large astroglial empty zone was observed in the CA1 region 2 days after SE. This astroglial loss was unrelated to vasogenic edema formation. There was no difference in SE-induced neuronal loss between saline- and mannitol-treated animals. Furthermore, mannitol treatment did not affect astroglial loss and vasogenic edema formation in the dentate gyrus and the piriform cortex. These findings suggest that mannitol treatment induces selective astroglial loss in the CA1 region independent of vasogenic edema formation following SE. These findings support the hypothesis that the susceptibility of astrocytes to SE is most likely due to the distinctive heterogeneity of astrocytes independent of hemodynamics. [BMB Reports 2015; 48(9): 507-512]     

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.     

Synthesis of new chiral peptide nucleic acid (PNA) monomers

We have synthesised a series of new chiral type I peptide nucleic acid monomers in total yields of 36-53%, derived from Val, Ile, Ser(Bzl), Pro, and Trp, employing convenient procedure.     

The presence of actin in nuclei: a critical appraisal.

To assess the significance of actin associations with nuclei, we have examined Amoeba proteus nuclei for the presence of labeled actin under a variety of circumstances without (in most instances) isolating nuclei or breaking up cytoplasms prior to the extraction of proteins.We first established that: the 42,000 dalton proteins (presumed to be actin) present in cytoplasm and non-isolated nuclei are identical electrophoretically; the putative actin of amebas has the same size and almost the same isoelectric point as rat muscle actin; and the peptide “fingerprints” of putative ameba actin and rat actin are very similar after tryptic digestion. We therefore concluded that the 42,000 dalton protein of ameba is actin.We determined that: the concentrations of actin in the cytoplasm and nucleus of amebas are the same; actin is readily lost from nuclei that are released from lysed cells; shortly after a ³⁵S-labeled nucleus is transplanted into unlabeled cytoplasm, or an unlabeled nucleus is transplanted into ³⁵S-labeled cytoplasm, the concentration of ³⁵S-actin in nucleus and cytoplasm is the same; and when cells containing ³⁵S-actin are subjected to long chase periods on unlabeled food, the concentrations of ³⁵S-actin in nucleus and cytoplasm fall in parallel. These observations taken together suggest that actin is not tightly associated with nuclei. Rather, actin may associate with nuclei for the trivial reason that the nuclear envelope is no barrier to free movement of that protein between the two compartments.We conclude that the mere presence of actin in nuclei is insufficient grounds for assuming that it has any role in nuclear functions, such as, for example, chromosome condensation.     

Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium

The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer.     

Involvement of IL-1 in the Maintenance of Masseter Muscle Activity and Glucose Homeostasis

Physical exercise reportedly stimulates IL-1 production within working skeletal muscles, but its physiological significance remains unknown due to the existence of two distinct IL-1 isoforms, IL-1α and IL-1β. The regulatory complexities of these two isoforms, in terms of which cells in muscles produce them and their distinct/redundant biological actions, have yet to be elucidated. Taking advantage of our masticatory behavior (Restrained/Gnawing) model, we herein show that IL-1α/1β-double-knockout (IL-1-KO) mice exhibit compromised masseter muscle (MM) activity which is at least partially attributable to abnormalities of glucose handling (rapid glycogen depletion along with impaired glucose uptake) and dysfunction of IL-6 upregulation in working MMs. In wild-type mice, masticatory behavior clearly increased IL-1β mRNA expression but no incremental protein abundance was detectable in whole MM homogenates, whereas immunohistochemical staining analysis revealed that both IL-1α- and IL-1β-immunopositive cells were recruited around blood vessels in the perimysium of MMs after masticatory behavior. In addition to the aforementioned phenotype of IL-1-KO mice, we found the IL-6 mRNA and protein levels in MMs after masticatory behavior to be significantly lower in IL-1-KO than in WT. Thus, our findings confirm that the locally-increased IL-1 elicited by masticatory behavior, although present small in amounts, contributes to supporting MM activity by maintaining normal glucose homeostasis in these muscles. Our data also underscore the importance of IL-1-mediated local interplay between autocrine myokines including IL-6 and paracrine cytokines in active skeletal muscles. This interplay is directly involved in MM performance and fatigability, perhaps mediated through maintaining muscular glucose homeostasis.