Ulinastatin attenuates protamine-induced cardiotoxicity in rats by inhibiting tumor necrosis factor alpha.

Protamine causes cardiac depression, which may be mediated by tumor necrosis factor alpha (TNF-α). Ulinastatin, a human urinary protease inhibitor, inhibits TNF-α. Here, we aimed to investigate whether ulinastatin prevented protamine-induced myocardial depression by inhibiting TNF-α. Rat hearts were perfused using a Langendorff system, and three protocols were followed. Protocol 1: The hearts were divided into saline, ulinastatin-low, and ulinastatin-high groups. Protamine was administered to each group, and myocardial contractility was the primary outcome. Protocol 2: The hearts were allotted to saline or ulinastatin group. Protamine was administered to each group. TNF-α expression in the coronary effluent and myocardial tissue was measured. Protocol 3: The hearts were allotted to saline and ulinastatin groups. Recombinant rat-TNF-α was administered to each group. Protamine alone reduced the maximum left ventricular pressure derivative (LV dP/dt max) by 45 ± 4%. In contrast, the reduction in LV dP/dt max was 4 ± 3% in the ulinastatin-high group. Compared with that in the saline group, the increase in TNF-α in the coronary effluent was attenuated in the ulinastatin group. Recombinant TNF-α alone reduced LV dP/dt max (- 21 ± 14%). In contrast, when TNF-α was added in the presence of ulinastatin, the decrease in LV dP/dt max was prevented significantly (- 3 ± 8%). We showed, for the first time, that ulinastatin protected against protamine-induced myocardial damage, both by inhibiting TNF-α synthesis and by directly preventing the cardiodepressant action of TNF-α.

Wilms' tumor gene WT1 promotes homologous recombination-mediated DNA damage repair.

The Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an oncogenic role in these malignancies. Alternative splicing at two sites yields four major isoforms, 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-), and all the isoforms are expressed in the malignancies. However, among the four isoforms, function of WT1[17AA(-)KTS(+)] isoform still remains undetermined. In the present study, we showed that forced expression of WT1[17AA(-)KTS(+)] isoform significantly inhibited apoptosis by DNA-damaging agents such as Doxorubicin, Mitomycin, Camptothesisn, and Bleomycin in immortalized fibroblast MRC5SV and cervical cancer HeLa cells. Knockdown of Rad51, an essential factor for homologous recombination (HR)-mediated DNA repair canceled the resistance to Doxorubicin induced by WT1[17AA(-)KTS(+)] isoform. GFP recombination assay showed that WT1[17AA(-)KTS(+)] isoform alone promoted HR, but that three other WT1 isoforms did not. WT1[17AA(-)KTS(+)] isoform significantly upregulated the expression of HR genes, XRCC2, Rad51D, and Rad54. Knockdown of XRCC2, Rad51D, and Rad54 inhibited the HR activity and canceled resistance to Doxorubicin in MRC5SV cells with forced expression of WT1[17AA(-)KTS(+)] isoform. Furthermore, chromatin immunoprecipitation (ChIP) assay showed the binding of WT1[17AA(-)KTS(+)] isoform protein to promoters of XRCC2 and Rad51D. Immunohistochemical study showed that Rad54 and XRCC2 proteins were highly expressed in the majority of non-small-cell lung cancer (NSCLC) and gastric cancer, and that expression of these two proteins was significantly correlated with that of WT1 protein in NSCLCs. Our results presented here showed that WT1[17AA(-)KTS(+)] isoform had a function to promote HR-mediated DNA repair.

Insulin preconditioning elevates p-Akt and cardiac contractility after reperfusion in the isolated ischemic rat heart.

Insulin induces cardioprotection partly via an antiapoptotic effect. However, the optimal timing of insulin administration for the best quality cardioprotection remains unclear. We tested the hypothesis that insulin administered prior to ischemia provides better cardioprotection than insulin administration after ischemia. Isolated rat hearts were prepared using Langendorff method and divided into three groups. The Pre-Ins group (Pre-Ins) received 0.5 U/L insulin prior to 15 min no-flow ischemia for 20 min followed by 20 min of reperfusion. The Post-Ins group (Post-Ins) received 0.5 U/L insulin during the reperfusion period only. The control group (Control) was perfused with KH buffer throughout. The maximum of left ventricular derivative of pressure development (dP/dt(max)) was recorded continuously. Measurements of TNF-α and p-Akt in each time point were assayed by ELISA. After reperfusion, dP/dt(max) in Pre-Ins was elevated, compared with Post-Ins at 10 minutes after reperfusion and Control at all-time points. TNF-α levels at 5 minutes after reperfusion in the Pre-Ins were lower than the others. After 5 minutes of reperfusion, p-Akt was elevated in Pre-Ins compared with the other groups. Insulin administration prior to ischemia provides better cardioprotection than insulin administration only at reperfusion. TNF-α suppression is possibly mediated via p-Akt leading to a reduction in contractile myocardial dysfunction.

[Effect of different preoperative rehydration on sugar metabolism; comparison between Arginaid Water and OS-1].

BACKGROUND: Enhanced Recovery After Surgery (ERAS) program recommends carbohydrate load before surgery. However, the dose and dosing method of carbohydrate load are not clear. In this paper, effect of different preoperative rehydration on sugar metabolism in healthy volunteers is reported. METHODS: Arginaid Water (ArgW) and OS-1 (OS) used as preoperative rehydration in Japan were employed for measuring sequential changes in sugar metabolism in blood. Both ArgW intake group and OS intake group started fasting at 9 PM. At 8 AM in the morning, respective preoperative rehydration 250 ml was taken as a bolus. Blood test was performed before intake, 30 minutes, 2 hours and 4 hours after intake. RESULTS: Subject included 10 healthy volunteers for ArgW and OS respectively. Subjects drank each preoperative rehydration two hours before entering operating room. In ArgW intake group, free fatty acid (FFA) and beta-hydroxybutyric acid concentration (beta-OHB) were reduced and sugar metabolism was favorably maintained. Meanwhile, OS intake group, FFA and beta-OHB were elevated and catabolism of adipose began. However, even if ArgW were taken, rebound increases of FFA and beta-OHB were observed after entering operating room. CONCLUSIONS: Optimum dosage and dosing method of preoperative carbohydrate should be scientifically verified in the future.

Peroxidase activity of DNA aptamer-pt complexes prepared with cisplatin.

DNA aptamers carrying Pt nanoparticles prepared with cisplatin showed peroxidase enzymatic activity while retaining the specific binding ability of the aptamers. Optimal preparation conditions of DNA-Pt complex prepared with cisplatin were investigated on the synthesis at pH 7-11, a reaction time of 1-18 h and 90 degrees C. The enzymatic reaction of DNA-Pt complex obeyed Michaelis-Menten kinetics. K(M) for the DNA-Pt complex was found to be of the same order as K(M) for hemin and hemin-DNA complex, but one order of magnitude higher than that of horseradish peroxidase. A sandwich type of DNA enzyme-linked aptamer assay (DLAA) using DNA-Pt complex successively detected target protein of thrombin. DLAA using DNA-Pt complex fractioned by ultrafiltration membranes having a molecular weight cut-off of 30 000 and 300 000 showed 1.9-times higher sensitivity than DLAA using DNA-Pt complex without fraction. The DNA-Pt complex having specific size was effective for the sensitive detection of thrombin in DLAA.

[A case of pneumothorax during laparoscopic surgery due to latent diaphragmatic defect].

A 38-year-old male healthy donor for renal transplantation was scheduled to undergo laparoscopic nephrectomy of the left kidney. After commencement of the surgery under general anesthesia, his vital signs were stable. When pneumoperitoneum was commenced using CO2, a rapid increase in the airway pressure was observed, and it became difficult to perform mechanical ventilation. After manual ventilation was initiated, the cause of the increased airway pressure was investigated. As a result, a defective pore, 3 cm in diameter, was confirmed in the left diaphragm and it was determined that pneumothorax developed from the pure CO2. A transient decrease in oxygen saturation was easily restored by manual ventilation. The blood pressure was relatively stable, and tension pneumothorax was not observed. For the defective pore in the diaphragm, endoscopic cerclage of the diaphragm was performed after insertion of a thoracostomy tube. Postoperative chest X-ray showed no signs of atelectasis, mediastinal emphysema, or aerodermectasia, suggesting the development of pneumothorax due to pure CO2. In this case, the defective pore in the diaphragm was caused accidentally by pneumoperitoneum, although the subject had had no prior symptoms. Latent diaphragmatic defect may be an important factor in pneumoperitoneum and other surgical procedures.

Preparation of a DNA aptamer-Pt complex and its use in the colorimetric sensing of thrombin and anti-thrombin antibodies.

DNA aptamers carrying Pt nanoparticles were prepared by the reaction of DNA aptamers (without functionalization with biotin, thiol, or other reactive groups) with K 2[PtCl 4] in solution at 60-90 degrees C. The DNA-Pt complexes possessed peroxidase enzymatic activity while retaining the specific binding ability of the aptamers. The enzymatic reaction of these complexes obeyed Michaelis-Menten kinetics. K M for the DNA-Pt complex was found to be on the same order as K M for hemin and hemin-DNA complex but 1 or 2 orders of magnitude higher than that of horseradish peroxidase. The rate of the reaction catalyzed by the DNA-Pt complex, k cat, was found to be on the same order as that of hemin and hemin-DNA complex but 2 or 3 orders of magnitude lower than that of horseradish peroxidase. Two types of DNAzyme-linked aptamer assays (DLAAs) were developed using these complexes, which successfully detected target proteins, with the sandwich type of DLAA targeting thrombin and the competitive type of DLAA targeting anti-thrombin IgA/G/M in serum. The DNA-Pt complexes retained their peroxidase enzymatic activity even after heat treatment. DLAAs having high thermal stability were developed using these complexes, which were free of animal and plant matter because neither antibodies nor horseradish peroxidase were used in their synthesis.

Preservation of hematopoietic stem and progenitor cells from umbilical cord blood stored in a surface derivatized with polymer nanosegments.

Tissue culture flasks were prepared with immobilized amphiphilic nanosegments of Pluronic F68 and F127, polyethylene oxide (PEO)-polypropylene oxide (PPO)-PEO triblock copolymers, on their surfaces. These so-called "Pluronic-immobilized flasks" were used for the preservation of hematopoietic stem and progenitor cells from umbilical cord blood. The expression ratio of surface markers (CD34) on hematopoietic stem and progenitor cells stored in Pluronic-immobilized flasks was significantly higher than that in polystyrene tissue culture flasks or commercially available bioinert flasks (i.e., low cell-binding cultureware). This was due to the presence of flexible brushlike segments of Pluronic on the Pluronic-immobilized flask. A good correlation was found between the number of CD34+ cells and the ratio of viable CD34+ cells from cord blood in several flasks after five days of storage. Therefore, the high number of CD34+ cells was thought to have originated from the high viability of these cells stored in Pluronic-immobilized flasks. It was found that there was an optimal surface concentration of Pluronic on the Pluronic-immobilized flask surfaces for the preservation (high number and survival) of these stem and progenitor cells. The foregoing results were attributable to the high density of Pluronic nanosegments on the flask surface, limiting the movement of these flexible segments.

Novel enzymatic properties of DNA-Pt complexes.

DNA-Pt complexes have shown novel enzymatic activity as a peroxidase similar to that of horseradish peroxidase in the colorimetric reaction with its substrate. The enzymatic activity of these complexes increased with increasing reaction time and pH in reaction solutions of DNA and K2[PtCl4]. This enhanced enzymatic activity was attributed to the increase in Pt conjugated to DNA in the complex. The enzymatic activity per unit mole of the DNA-Pt complex was significantly higher for complexes prepared with high molecular weight DNA because the enzymatic activity of the complex per repeat unit of DNA was almost constant for these complexes prepared under the same reaction conditions. All the DNA-Pt complexes in this study prepared with different DNA sequences (i.e., [A]20, [G]20, [C]20, [T]20, and [AG]10) exhibited peroxidase enzymatic activity. These complexes showed good thermal stability as compared to native horseradish peroxidase.

Temperature-induced cell detachment on immobilized pluronic surface.

The Pluronic F68 and F127, a triblock copolymer of ethylene oxide and propylene oxide, was activated using carbonyldiimidazole (CDI), and CDI-activated Pluronic F68 and F127 was subsequently immobilized on the surface of a poly-L-lysine-coated polystyrene tissue culture flask. Cell culture was performed on the Pluronic-immobilized flask. The morphology of fibroblasts (L929 cells) on the Pluronic F127-immobilized flask was mainly spherical, and showed less spreading behavior than that on the Pluronic F68-immobilized flask and conventional tissue culture flask. This observation indicates that L929 cells on Pluronic F127-immobilized flasks were cultured in a bio-inert environment. L929 cells were successively detached from both Pluronic F127-immobilized flask and Pluronic F68-immobilized flask by cooling the flask to 4-15 degrees C. This detachment is due to the hydration and dehydration properties of Pluronic, depending on the temperature. Umbilical cord blood was cultured in the Pluronic F127-immobilized and conventional polystyrene tissue culture flasks at 37 degrees C. The expression ratio of surface markers on hematopoietic stem cells (CD34 and CD133) cultured in the Pluronic F127-immobilized flask was significantly higher than that of the cells in polystyrene tissue culture flask.

Clonidine-ephedrine combination reduces pain on injection of propofol and blunts hemodynamic stress responses during the induction sequence.

STUDY OBJECTIVES: To evaluate the effects of clonidine and ephedrine on propofol-induced pain and on hemodynamic changes during the induction sequence. DESIGN: This was a prospective, randomized, double-blind study. SETTING: The study was conducted at a university hospital. PATIENTS: 200 ASA physical status I or II adult patients scheduled for elective surgery. INTERVENTIONS: Patients were randomly allocated to one of 4 groups (50 patients per group): clonidine-ephedrine (CE), clonidine-saline (CS), diazepam-ephedrine (DE), and diazepam-saline (DS). Thirty seconds after the administration of ephedrine or saline, propofol 2 mg/kg was infused at a rate of 18.3 mL/min. MEASUREMENTS: Patients were asked whether they had pain due to propofol injection. A blinded investigator evaluated the pain score: 0 = no pain, 1 = mild pain, 2 = severe pain without behavioral signs such as grimace or arm withdrawal movement, and 3 = severe pain accompanied by behavioral signs. Mean arterial blood pressure (MAP) and heart rate (HR) were measured at 1-minute intervals from just before the administration of ephedrine or saline to 5 minutes after the tracheal intubation. MAIN RESULTS: Median pain score in CE was significantly lower than those in the other groups (P < 0.0001). Pain scores in CS and DE were significantly lower than that in DS (P < 0.05). Ephedrine increased HR in CE and DE (P < 0.05), but clonidine did not augment the effect. Mean arterial blood pressure before tracheal intubation decreased to comparable values in all groups. After the intubation, mean arterial blood pressure and HR in CE and CS were significantly lower than those in DE and DS (P < 0.05). CONCLUSIONS: Combination of clonidine and ephedrine effectively reduced propofol-induced pain, but did not prevent propofol-induced hypotension. Clonidine did not augment low dose of ephedrine-induced increase in HR and produced stable hemodynamic condition during the induction sequence.

Bioinert surface of pluronic-immobilized flask for preservation of hematopoietic stem cells.

The bioinert materials on which cells do not proliferate, differentiate, nor de-differentiate should be useful for the culture and preservation of stem cells. The Pluronic F127, a triblock copolymer of ethylene oxide, and propylene oxide was activated using carbonyldiimidazole (CDI), and CDI-activated Pluronic was subsequently immobilized on the surface of a lysine-coated polystyrene tissue culture flask. The morphology of fibroblasts (L929 cells) on the Pluronic-immobilized flask was spherical, and did not show spreading behavior. This observation indicates that L929 cells on the Pluronic-immobilized flask were cultured in a bioinert environment. The expression ratio of surface markers on hematopoietic stem cells (CD34 and CD133) cultured in the Pluronic-immobilized flask was significantly higher than that in polystyrene tissue culture flask and commercially available bioinert flask (i.e., low cell binding cultureware). This is caused by the existence of hydrophilic segments of Pluronic F127 on the Pluronic-immobilized flask.

[Refractory generalized convulsions in a patient undergoing brain tumor resection during propofol anesthesia].

Propofol has been used to treat convulsions, while the drug is known to induce convulsions. We described a case of generalized convulsions during brain tumor resection under propofol anesthesia. A 24-year-old man was scheduled to undergo brain tumor resection. He had no history of epilepsy. Anesthesia was induced and maintained with propofol and fentanyl. During the craniotomy, the patient developed generalized convulsions. Diazepam, thiamylal, and phenytoin were given intravenously and the seizure activity resolved. Generalized convulsions recurred three times during the operation. Postoperative course was uneventful. On the 16 th postoperative day, the patient underwent ventriculoperitoneal shunt under general anesthesia using sevoflurane, nitrous oxide and oxygen. Convulsions were not noted intra- and postoperatively. Because convulsions did not occur during sevoflurane anesthesia and the patient had no history of epilepsy, propofol may have induced a generalized convulsions on the first operation.