Relationship between perioperative cardiopulmonary bypass time, platelet count, fibrinogen level, rotational thromboelastometry data, antithrombin level, and blood loss volume and the effects of deep hypothermic circulatory arrest: An observational study.

INTRODUCTION: We hypothesized that perioperative fibrinogen level, platelet count, and rotational thromboelastometry (ROTEM) data values decrease in proportion to cardiopulmonary bypass (CPB) time, particularly in patients who underwent deep hypothermic circulatory arrest (DHCA). METHODS: A total of 160 patients were enrolled and divided into the following three groups depending on CPB time: <2-h, 2- 3-h, and >3-h groups. Blood samples were obtained during CPB weaning. Platelet count, ROTEM data, fibrinogen level, and antithrombin level were determined. For propensity matching, we selected 15 patients who underwent DHCA and 15 patients who did not undergo DHCA and used propensity scores to match CPB time and other characteristics. RESULTS: The <2-h, 2-3-h, and >3-h groups included 74, 63, and 23 patients, respectively. No significant differences in platelet count and fibrinogen level were observed between the groups. Antithrombin level and amplitude of clot firmness at 10 min in the EXTEM and FIBTEM tests were lowest in the >3-h group. Similarly, blood loss volume and transfusion volume were highest in the >3-h group. Significant differences in platelet count, ROTEM data, lowest esophageal and bladder temperatures, and transfusion volume were observed between patients who underwent DHCA and patients who did not undergo DHCA. CONCLUSIONS: The longer the CPB time, the greater the perioperative blood loss volume and transfusion volume, particularly if CPB time is greater than 3 hours. Sub-group analysis revealed that DHCA affects perioperative platelet count and function as well as blood loss volume.

A toxicological evaluation of a claudin modulator, the C-terminal fragment of Clostridium perfringens enterotoxin, in mice.

Tight junctions (TJs) maintain cellular polarity between the apical and basolateral region of epithelial cells. Claudin, a tetra-transmembrane protein, plays a pivotal role in the barrier function of TJs. We previously found that a claudin modulator, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), may be a promising candidate for improving the mucosal absorption of drugs. C-CPE is a fragment of enterotoxin, and putative CPE claudin receptors are highly expressed in liver and kidney. The safety and antigenicity of C-CPE must be evaluated for future clinical application. Therefore, we evaluated whether C-CPE administration in mice leads to tissue injury or production of antibodies. Intravenous administration of C-CPE at 5 mg/kg, which is a more than 25-fold higher dose than that used in a murine mucosal absorption model, did not increase biochemical markers of liver and kidney injury even after 11 injections once a week. Nasal C-CPE administration (2 mg/kg) once a week for 11 administrations also did not increase these biochemical markers, but 6 administrations of C-CPE resulted in elevation of C-CPE-specific serum IgG. These results indicate that development of a less antigenic claudin modulator will be essential for future clinical application of a C-CPE-based mucosal absorption enhancer.

Effect of nitrous oxide on propofol requirement during target-controlled infusion for oocyte retrieval.

BACKGROUND: Oocyte (egg) retrieval for in-vitro fertilization is a relatively short procedure, usually performed as an outpatient. Propofol is a suitable anesthetic agent. Target-controlled infusion is a recently developed system that aids rapid recovery from propofol anesthesia. This study sought to determine the target concentration of propofol required to prevent movement in 50% (Cp50) and 95% (Cp95) of women during oocyte retrieval, and investigated whether supplemental nitrous oxide (N2O) modified the Cp50 and Cp95. METHODS: Forty-seven women scheduled for oocyte retrieval were randomly selected to receive either O2-air mixture (control group; n = 23) or 50% O2-N2O mixture (Nitrous oxide group; n = 24). Propofol was infused using a target-controlled infusion system with the concentration determined by up-down sequential allocation using 0.5 microg/mL step size. Transvaginal oocyte retrieval was performed after reaching target blood concentration. Patient responses to oocyte retrieval were noted as either no movement or movement. RESULTS: Using target-controlled infusion the Cp50 was 4.1 microg/mL in the control group and 3.3 microg/mL in the nitrous oxide group. Calculated Cp95 values were 4.0 microg/mL and 5.1 microg/mL with and without 50% nitrous oxide respectively. CONCLUSIONS: The Cp50 value for target-controlled infusion propofol during oocyte retrieval was significantly reduced by a factor of 1.24 (95% CI 1.07-1.44) with the use of 50% nitrous oxide.

Specific binding of amyloid-beta-protein to IMR-32 neuroblastoma cell membrane.

In flow cytometry using two detecting methods, we have found that amyloid-beta-protein(1-40) [Abeta(1-40)] has high affinity to IMR-32 neuroblastoma cell membrane when it is aggregated to form beta-sheet conformation, whereas random coil small Abeta-species has low affinity. The difference in the binding ability to the cell membranes well accounts for the cytotoxicity of Abeta(1-40); namely, aggregated beta-sheet Abeta(1-40) gives cytotoxicity higher than random coil Abeta(1-40). Specific binding between Abeta(1-40) and ganglioside GM1 of the raft-like domain in lipid membrane is suggested from a surface plasmon resonance (SPR) experiment.

Relation between fentanyl dose and predicted EC50 of propofol for laryngeal mask insertion.

BACKGROUND: This study sought to determine the effective concentration for 50% of the attempts to secure laryngeal mask insertion (predicted EC(50LMA)) of propofol using a target-controlled infusion (Diprifusor) and investigated whether fentanyl influenced these required concentrations, respiratory rate (RR) and bispectral index (BIS). METHODS: Sixty-four elective unpremedicated patients were randomly assigned to four groups (n = 16 for each group) and given saline (control) or fentanyl 0.5, 1 or 2 micro g kg(-1). Propofol target concentration was determined by a modification of Dixon's up-and-down method. Laryngeal mask airway insertion was attempted without neuromuscular blocking drugs after equilibration had been established for >10 min. Movement was defined as presence of bucking or gross purposeful muscular movement within 1 min after insertion. EC(50LMA) values were obtained by calculating the mean of 16 patients in each group. RESULTS: Predicted EC(50LMA) of the control, fentanyl 0.5, 1 and 2 micro g kg(-1) groups were 3.25 (0.20), 2.06 (0.55), 1.69 (0.38) and 1.50 (0.54) micro g ml(-1) respectively; those of all fentanyl groups were significantly lower than that of control. RR was decreased in relation to the fentanyl dose up to 1 micro g kg(-1). BIS values after fentanyl 1 and 2 micro g kg(-1) were significantly greater than in the control and 0.5 micro g kg(-1) groups. CONCLUSIONS: A fentanyl dose of 0.5 micro g kg(-1) is sufficient to decrease predicted EC(50LMA) with minimum respiratory depression and without a high BIS value.

Predicted values of propofol EC50 and sevoflurane concentration for insertion of laryngeal mask Classic and ProSeal.

BACKGROUND: A new laryngeal mask airway, the ProSeal (PLMA), is said to be more difficult to insert than the laryngeal mask airway Classic (CLMA) using propofol anaesthesia. Therefore, we expected a greater dose of propofol and sevoflurane to be required to insert the PLMA compared with the CLMA. We determined the effective concentration 50% (EC(50)) of propofol and end-tidal sevoflurane to allow insertion of the PLMA and the CLMA. METHODS: Seventy-six elective female patients (aged 20-60 yr and ASA I-II) were randomly assigned to one of four groups. Either a PLMA or a CLMA was inserted using either propofol target controlled infusion or sevoflurane. Both propofol and sevoflurane targets were determined with a modified Dixon's up-and-down method. After equilibration between the predetermined blood and effect site concentrations, which had been held steady for more than 10 min, LMA insertion was attempted without neuromuscular block. RESULTS: The predicted EC(50CLMA) and EC(50PLMA) for propofol were 3.14 (0.33) and 4.32 (0.67) micro g ml(-1). E'(CLMA) and E'(PLMA) of sevoflurane (mean (SD)) were 2.36 (0.22) and 2.82 (0.45)% (P<0.01 and 0.05, respectively). CONCLUSIONS: The estimated concentration of propofol and the sevoflurane concentration needed to allow insertion of the ProSeal are respectively 38 and 20% greater than those needed for insertion of the Classic LMA.

Practical assay and molecular mechanism of aggregation inhibitors of beta-amyloid.

Beta-Amyloid peptide (Abeta) is the main protein component of neuritic plaques in the brain of patients of Alzheimer's disease (AD), and its neurotoxicity would be exposed by the formation of aggregates. The aggregation inhibitors composed of an Abeta recognition element (KLVFF) and a hydrophilic moiety are evaluated by a novel fluorescence assay. These compounds inhibit growth of the model aggregates on the KLVFF immobilized surface. In addition, some compounds also possess disrupting activities of preformed aggregates. These compounds could be a key candidate for therapeutic drugs for AD by their novel molecular mechanisms.

Cp50 of propofol for laryngeal mask airway insertion using predicted concentrations with and without nitrous oxide.

This study sought to determine the predicted Cp50 of propofol required for laryngeal mask airway insertion (Cp50LMA) and to investigate whether nitrous oxide reduces these required concentrations. Using target-controlled infusion and incorporating the standard Diprifusor pharmacokinetic model, 46 unpremedicated patients were randomly assigned to one of two groups. The patients received either 40% oxygen in air (control group: n = 23), or 60% nitrous oxide in oxygen (nitrous oxide group: n = 23). The target concentration for each patient was determined using the up and down method. Following equilibration between the predetermined blood and effect site concentrations, had been established for > 10 min, laryngeal mask airway insertion was attempted without neuromuscular relaxants. The data were analysed using a probit analysis to obtain Cp50LMA levels. The values for Cp50LMA were 3.24 micro g.ml-1 in the control group and 1.93 microg.ml-1 in the nitrous oxide group.

Mononuclear platinum(II) complex with 2-phenylpyridine ligands showing high cytotoxicity against mouse sarcoma 180 cells acquiring high cisplatin resistance.

Mouse sarcoma 180 cell with a 25-fold higher cisplatin (CDDP) resistance, termed S-180cisR, is newly established. S-180cisR cells grow quite slowly in the presence of CDDP with high concentration. This may show that S-180cisR cells modulate the cell cycle to acquire CDDP resistance. P-Glycoprotein is selectively expressed on the surface of S-180cisR, which is not on CDDP-sensitive S-180 parent cells. In an experiment using an inhibitor (verapamil) of P-glycoprotein, cytotoxicity of CDDP against S-180cisR is significantly increased (viz., IC(50) value is decreased) and accumulation of CDDP in S-180cisR cells is also increased. These results indicate that enhanced pumping-out of CDDP by P-glycoprotein should be one of the reasons for the CDDP resistance of S-180cisR. A platinum(II) complex with a cyclometalated 2-phenylpyridine ligand and a nonchelated one (complex 5) is synthesized, and its structure is determined by X-ray structural analysis. Complex 5 has a cyctotoxicity against S-180cisR higher than that of CDDP and its derivatives with 2- or 3-substituted pyridine ligands (complexes 2-4, 6, 7). Complex 5 is incorporated in S-180cisR to an enormously greater extent than CDDP; that is, the ratio of accumulated platinum amount after 3 h is 61.9. In S-180 parent cells, on the other hand, the ratio remains 8.1. This high accumulation of complex 5 into S-180cisR must account for the higher activity of complex 5 against S-180cisR compared to CDDP.

Identification of the molecular interaction site of amyloid beta peptide by using a fluorescence assay.

Beta-amyloid peptides (Abeta) are the main protein components of neuritic plaques and are important in the pathogenesis of Alzheimer's disease. It is reported that Abeta itself is not toxic; however, it becomes toxic to neuronal cells once it has aggregated into amyloid fibrils by peptide-peptide interactions. In this study, to specify the molecular mechanism of aggregation, a novel fluorescence assay was designed. For this purpose, possible partial peptides (38 types of 5-mer) were synthesized on solid-phase. The molecular interactions were examined by a fluorescence probe possessing Lys-Leu-Val-Phe-Phe (KLVFF) as a molecular recognition site. KLVFF is known to be a minimum sequence for formation of the Abeta aggregate. A specific interaction was observed between labeled and immobilized KLVFF. It suggests that the aggregation of Abeta was controlled by the recognition of KLVFF itself by hydrophobic and electrostatic interactions.

Platinum (II) complex with cyclometalating 2-phenylpyridine ligand showing high cytotoxicity against cisplatin-resistant cell.

A platinum (II) mononuclear complex with two kinds of 2-phenylpyridine which coordinate as cyclometalated and non-chelated ligands shows high cytotoxicity against cisplatin-resistant mouse sarcoma 180 cell in comparison with its related complexes.

[Anesthesia for emergency surgery in 2 extremely low birth weight infants with ileus].

Two extremely-low-birth-weight infants, weighing each 684 and 975 g at birth, underwent emergency surgery because of ileus. Our previous experience with an extremely low birth weight infant, whose hemodynamic control during the surgery had been difficult without administering extra preoperative fluid and transfusion, made us administer enough fluid and transfusion during operation although their urine output was more than 2 ml.kg-1.hr-1. We gave intravenous volume to the present 2 cases before the operations despite the level of preoperative urine output and made their hemodynamic situation more stable during surgeries. We conclude it is very important to administer some amounts of intravenous volume (approximately 8-12 ml.kg-1.hr-1) in extremely low birth weight infants for emergency surgery with ileus regardless of their preoperative urine output.

Preparation of functional liposomes with peptide ligands and their binding to cell membranes.

Two novel lipopeptides, which have the peptide ligands [alpha-melanocyte stimulating hormone (alpha-MSH)] sequence and repeated [Gly-Arg-Gly-Asp-Se (GRGDS) sequence], are designed, synthesized by the solid-phase method, and introduced into liposome membranes by the freeze-thaw method. These liposomes bearing the peptide ligands on their surface are expected to bind to cell membranes. We have confirmed that the lipopeptides are introduced into liposome membranes almost quantitatively, while such a high degree of incorporation has not been accomplished in conventional methods. In this respect, the present method is superior to prepare surface-modified liposomes that are applicable to drug carriers and so on. We have also confirmed by using immunoelectron microscopy that the peptide ligands are actually located in an aqueous phase. It has been shown by flow cytometry that the liposome bearing alpha-MSH peptide ligand binds to B16 cells and the liposome bearing the repeated GRGDS sequence binds to NIH3T3 cells.

Lipid analog with 2-nitrophenol trigger designed for liposome fusion at physiological pH.

A novel lipid analog with two long alkyl (C16) chains, an aspartate skeleton, a connecting alkyl (C8) chain, and 2-nitrophenol trigger group is synthesized by an efficient synthetic route, which can induce liposome fusion at physiological pH.

[Depressive effects of propofol on apoptotic injury and delayed neuronal death after forebrain ischemia in the rat--comparison with nitrous oxide-oxygen-isoflurane].

We investigated the brain protection effects of propofol anesthesia and nitrous oxide-oxygen-isoflurane anesthesia (GOI) using forebrain ischemic model of male Sprague-Dawley rats. Propofol group (P, n = 15) was anesthetized with propofol, oxygen and nitrogen (FIO2 = 0.33), and isoflurane group (GOI, n = 15) with 66% nitrous oxide, 33% oxygen and 1.2% isoflurane under mechanical ventilation. The anesthesia was deepened until electroencephalographic burst suppression appeared in each group. The bilateral common carotid arteries were, then, occluded for 10 minutes while the blood pressure was maintained at about 40 mmHg by venesection. The venesected blood was returned at the end of ischemic period. The animals were kept and fed in cage after emergence. On the day 2, 4, and 7, five animals of each group were sacrificed and the microscopic samples were obtained. The CA-1 cells of hippocampus were then stained with hematoxylin and eosin for the delayed neuronal death (DND) and with TUNEL method for the apoptosis. Propofol reduced the apoptosis, i.e., reduced the TUNEL positive cell count (GOI = 121.2 +/- 25.2.mm-1; P = 53.8 +/- 11.4.mm-1; P < 0.01; mean +/- SD) on the day 2 after ischemia, and also reduced the delayed neuronal death (alive CA-1 cell count; GOI = 18.1 +/- 8.9.mm-1; P = 33.1 +/- 12.8.mm-1; P < 0.01) on the day 7 after ischemia. It is important to determine the recovery interval after brain ischemia in detection of DND and apoptosis. We conclude that propofol inhibits neuronal apoptosis after brain ischemia and consequently reduces the delayed neuronal death in the CA-1 pyramidal cell layer of the hippocampus.

Selective synthesis and kinetic measurement of 1:1 and 2:2 cyclic compounds containing 1,4,7,10-tetraazacyclododecane and azobenzene units

1:1 cyclic compounds 8a-c (51-55%) and 2:2 cyclic compounds 9a-c (20-49%) containing 1,4,7,10-tetraazacyclododecane (cyclen) and azobenzene units were selectively synthesized under UV irradiation (330 nm < lambda < 380 nm) and in the dark. Synthesis depended on the wavelength of irradiation light and the length of methylene chains of the linker between the cyclen and azobenzene units. A study of NMR and UV-vis spectra indicated that properties of 8a-c and 9a-c are closely related to their structural flexibility. Rate constants (k) and thermodynamic parameters (DeltaG(), DeltaH(), and DeltaS()) of 8a-c and 9a-c were studied in nonpolar media (benzene) and polar media (methanol). The cis to trans isomerization rates in the dark for these cyclic compounds increase with ring size or structural flexibility (8a < 8c < 8b < 9a < 9b < 9c). In principle, DeltaS() dominates DeltaG() in cyclic compounds.

Synthesis of a novel lipopeptide with alpha-melanocyte-stimulating hormone peptide ligand and its effect on liposome stability.

Introduction of liposomes into target cells is important for drug delivery systems. For this purpose, the surface of the liposome is equipped with ligand peptides, which may bind to specific receptors on the cell membrane. An artificial novel lipopeptide (MSH-C4A2) containing the alpha-melanocyte-stimulating hormone (alpha-MSH) sequence and two long alkyl chains was designed and synthesized, and the liposome, composed of egg phosphatidylcholine (EPC) and MSH-C4A2, was prepared. The stability of the liposome was estimated by measuring calcein leakage from the liposome inner phase. The stability of the liposome decreased upon addition of MSH-A4C2, which seemed to be attributable to the amphiphilic property of the peptide moiety (alpha-MSH) of MSH-A2C4. The stability was, however, recovered fairly well upon addition of cholesterol (Ch) or phosphatidylglycerol (PG). It was concluded therefore that the ternary system, MSH-C4A2/Ch/EPC or MSH-C4A2/PG/EPC, is suitable for preparing the functional liposome.

Significance of VLA-4-VCAM-1 interaction and CD44 for transendothelial invasion in a bone marrow metastatic myeloma model.

In previous work, we established the B9/BM1 syngeneic murine bone marrow metastasis model. Interleukin (IL)-6-dependent. IL-1-producing B9/BM1 cells, which colonize the vertebral and femoral marrow after i.v. injection, show great similarity in cell surface phenotype to human myeloma cells, especially the expression of 3 adhesion molecules, CD44, VLA-4 and ICAM-1. Here we investigated the function of these adhesion molecules by binding and transendothelial invasion assays using a newly established bone marrow-derived endothelial cell line (BMEC). A combination of monoclonal antibodies against CD44 and VLA-4 significantly inhibited the adherence of B9/BM1 cells to BMEC and anti-CD44 mAb especially blocked B9/BM1 transendothelial invasion of unstimulated BMEC cells. Results of additional experiments, in which the cells were treated with anti-CD44 and hyaluronidase, demonstrated that the interaction of CD44 molecules on B9/BM1 cells with hyaluronan on BMEC cells was a critical factor in both adhesion and transendothelial invasion in this model. However, stimulation of BMEC with TNFalpha resulted in increased invasion by B9/BM1 cells, which was completely suppressed by anti-VCAM-1 mAb, implicating a significant role of this adhesion molecule in this process during inflammation.

Enhancement of type IV collagenases by highly metastatic variants of HT1080 fibrosarcoma cells established by a transendothelial invasion system in vitro.

A novel in vitro invasion assay system was established in this laboratory, in which the invasion of tumor cells after interaction with endothelial cells could be examined. Two variant cell lines (FP-10, FP-21) were established from parental HT1080 cells using this assay system. FP-10 and FP-21 cells had higher invasive and metastatic potential than the parental cells both in vitro and in vivo. The activity of anchorage-independent proliferation and the adhesion to the HUVEC monolayer of FP-10 and FP-21 was greater than the parental cells. The secretion of type IV collagenase (both MMP-2 and MMP-9) was also increased more significantly by the variant cells than by the parental cells, and the expression of uPA mRNA was higher in FP-10 and FP-21. Treatment of variant cells with human TIMP-2 remarkably suppressed the increment of the in vitro invasion to the same level as parental cells. These results suggest that this in vitro transendothelial invasion system accelerates multiple mechanisms of the metastasis by HT1080, especially the production of type IV collagenases. It can thus provide a useful model of tumor metastasis.