Endothelial NO Synthase (eNOS) phosphorylation regulates coronary diameter during ischemia-reperfusion in association with oxidative stress

The link between endothelial nitric oxide synthase (eNOS) activation and vascular diameter during ischemia-reperfusion was investigated in the rat heart. After short (<30 min) and long (>45 min) time of ischemia conferred by coronary artery occlusion of the rats, reperfusion caused dilatation and constriction of arterioles, respectively. Partial oxygen pressure (pO2) measurement of the heart by the electrode confirmed the hyper-perfusion and no-reflow phenomena during reperfusion, as well as myocardial ischemia. The vascular diameter was correlated with phosphorylation of Akt and serine 1177 residue of eNOS, and formation of NO-bound guanylate cyclase (GC) by immuoflorescence study. Western blotting confirmed the phosphorylation of eNOS-Ser1177 depending on ischemia time. The constriction during reperfusion after 45 min of ischemia is supposedly caused by the inhibition of Akt-mediated eNOS-Ser1177 phosphorylation, which was suppressed by a PKC inhibitor chelerythrine, or ROS scavengers N-2-mercaptopropionyl glycine (MPG) and 4,5-Dihydroxy-1, 3-benzenedisulfonic acid disodium salt (Tiron). However, an endothelin receptor antagonist BQ123 alleviated the vasoconstriction by increasing NO availability but not eNOS-Ser1177 phosphorylation. Thus, vascular patency is correlated with eNOS-Ser1177 phosphorylation in association with ROS, and PKC during reperfusion. Endothelin inhibits vasodilatation by reducing NO availability during reperfusion.     

N-acetyl-l-methionine is a superior protectant of human serum albumin against photo-oxidation and reactive oxygen species compared to N-acetyl-l-tryptophan

Background

Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products.

Methods

Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4 weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration.

Results

N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present.

Conclusion

The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations.

General significance

N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.     

Identification of ATP-sensitive Potassium Channel in Frog Ventricular Myocytes

Nuclear magnetic resonance (NMR) microimaging and proton relaxation times were used to monitor differences between the hydration state of the nucleus and cytoplasm in the Rana pipiens oocyte. Individual isolated ovarian oocytes were imaged in a drop of Ringer's solution with an in-plane resolution of 80 μm. Proton spin echo images of oocytes arrested in prophase I indicated a marked difference in contrast between nucleoplasm and cytoplasm with additional intensity gradations between the yolk platelet-rich region of the cytoplasm and regions with little yolk. Neither shortening τ_e (spin echo time) to 9 msec (from 18 msec) nor lengthening τ_r (spin recovery time) to 2 sec (from 0.5 sec) reduced the observed contrast between nucleus and cytoplasm. Water proton T₁ (spin-lattice) relaxation times of oocyte suspensions indicated three water compartments that corresponded to extracellular medium (T₁= 3.0 sec), cytoplasm (T₁= 0.8 sec) and nucleoplasm (T₁= 1.6 sec). The 1.6 sec compartment disappeared at the time of nuclear breakdown. Measurements of plasma and nuclear membrane potentials with KCl-filled glass microelectrodes demonstrated that the prophase I oocyte nucleus was about 25 mV inside positive relative to the extracellular medium. A model for the prophase-arrested oocyte is proposed in which a high concentration of large impermeant ions together with small counter ions set up a Donnan-type equilibrium that results in an increased distribution of water within the nucleus in comparison with the cytosol. This study indicates: (i) a slow exchange between two or more intracellular water compartments on the NMR time-scale, (ii) an increased rotational correlation time for water molecules in both the cytoplasmic and nuclear compartments compared to bulk water, and (iii) a higher water content (per unit dry mass) of the nucleus compared to the cytoplasm, and (iv) the existence of a large (about 75 mV positive) electropotential difference between the nuclear and cytoplasmic compartments. Received: 18 January 1996/Revised: 29 April 1996     

Temperature- and growth-phase-dependent changes in membrane fatty acid compositions of Brevibacterium ammoniagenes

Fatty acids newly synthesized by Brevibacterium ammoniagenes grown at different temperatures were analyzed. The assay temperature, not the growth temperature, was found to be the major factor affecting the unsaturated/saturated ratio of newly synthesized fatty acids in logarithmic-phase cells. However, in the stationary-phase cells the growth temperature also affected the product profile significantly; cells grown at 7 degrees C produced relatively more oleate and stearate and less palmitate and hexadecenoate when shifted up to 37 degrees C than did cells grown and assayed at 37 degrees C. The unsaturated/saturated ratio as well as average chain length of fatty acids also varied along with the progress of isothermal growth phase. These changes in fatty acid product profiles observed in vivo could be mimicked in vitro assays of the fatty acid synthetase by changing malonyl-CoA concentrations. Our results suggest that the malonyl-CoA concentration is a factor which, in addition to temperature, determines growth-phase-dependent and growth-temperature-dependent changes in the unsaturated/saturated ratios of fatty acids.     

Ramicandelaber, a new genus of the Kickxellales, Zygomycetes

Ramicandelaber, a new genus of Zygomycetes is erected to accommodateRamicandelaber longisporus sp. nov. The fungus has hyphal septa with median plugs and forms homologous structures to sporocladia and pseudophialides. These characteristics suggest that it belongs to the Kickxellales, Zygomycetes.     

Injection of the insulin receptor alpha subunit increases blood glucose levels in mice

Using the expression vector of the truncated human insulin receptor (hIR), we have constructed a stable Chinese hamster ovary (CHO) cell line which secretes the His-tagged alpha subunit (insulin-binding domain) of hIR into medium. To examine characteristics of the His-tagged hIRalpha, we purified the protein secreted from the CHO cells. The His-tagged hIRalpha was glycosylated and processed a dimer. The molecule bound insulin with an affinity similar to that of the intact hIR. The His-tagged full length of hIR was autophosphorylated by insulin stimulation in CHO cells. Injection of the purified His-tagged hIRalpha into veins of mice increased in the concentration of blood glucose within 30 min. The intraperitoneal glucose tolerance test (ipGTT) done after injection of the purified His-tagged hIRalpha showed evidence of a marked hyperglycemia. These findings provide direct evidence that the presence of hIRalpha in the blood stream inhibits insulin actions by binding with plasma insulin.     

Constitutive Turnover of Phosphorylation at Thr-412 of Human p57/Coronin-1 Regulates the Interaction with Actin

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/coronin-1 regulates its interaction with actin.     

A simple method for isolation of nuclei from <Emphasis Type="Italic">Basidiobolus ranarum</Emphasis> (Zygomycota)

A simple method for isolating the nuclei from Basidiobolus ranarum was established. To improve the yield and purity of nuclei, we investigated maceration methods, buffer composition, and centrifugation conditions to establish an optimal procedure. Basidiobolus ranarum cultured for 5 days was enzymatically macerated and then homogenized and filtrated through stainless steel sieves. The crude cell homogenate was loaded on a layer of buffer containing 50% glycerol and centrifuged at 1500 g. The resultant pellet contained pure nuclei.