PMEA polymer-coated PVC tubing maintains anti-thrombogenic properties during in vitro whole blood circulation.

Poly(2-methoxyethylacrylate) (PMEA) is a new coating material that appears to reduce protein and platelet adsorption. However, the exact performance of PMEA coated circuit remains to be revealed in well-controlled experiments. Therefore, we compared its hemocompatibility with covalent-bound heparin-, and non-coated circuits during 6 hours of in vitro circulation, using donor blood from six volunteers. In our model, simple tubing circuits containing one-way ball valve were placed on the rotary table, which moved alternatively to generate pulsatile recirculation of heparinized human blood inside the tubing. Using this model, we expected fine assessment of the material surface, because we could reduce blood damage by avoiding air and a blood pump. Moreover, the small capacity of circuit allowed us to compare three kinds of circuits using a single unit of donor blood, eliminating effects by possible variations between blood donors. The anti-thrombin capacity of the PMEA-coated circuits was maintained even after six hours blood circulation, whereas surface thrombin generation increased markedly after use in non-coated circuits (P<0.05). Deposition of fibrin onto PMEA circuits was reduced more than 30% compared with heparin and non-coated circuits (P<0.05). However, the increase of plasma Factor XIIa was similar in all circuits. Increase of CD11b expression on circulating leukocytes and of plasma C3a was ameliorated in the heparin- and PMEA-coated circuits (P<0.05). PMEA-coated circuits appear to maintain their anti-thrombogenicity during use, otherwise PMEA-coated and heparin-coated circuits showed a similar character in hemocompatibility. This long-standing anti-thrombogenicity might be attributable to less adsorption of activated blood components onto the surface.

The mitochondrial genomes of soft ticks have an arrangement of genes that has remained unchanged for over 400 million years.

There are two major groups of ticks: soft ticks and hard ticks. The hard ticks comprise the prostriate ticks and the metastriate ticks. The mitochondrial (mt) genomes of one species of prostriate tick and two species of metastriate ticks had been sequenced prior to our study. The prostriate tick has the ancestral arrangement of mt genes of arthropods, whereas the two metastriate ticks have rearrangements of eight genes and duplicate control regions. However, the arrangement of genes in the mt genomes of soft ticks had not been studied. We sequenced the mt genomes of two species of soft ticks, Carios capensis and Ornithodoros moubata, and a metastriate tick, Haemaphysalis flava. We found that the soft ticks have the ancestral arrangement of mt genes of arthropods, whereas the metastriate tick, H. flava, shares the rearrangements of mt genes and duplicate control regions with the other two metastriate ticks that have previously been studied. Our study indicates that gene rearrangements and duplicate control regions in mt genomes occurred once in the most recent common ancestor of metastriate ticks, whereas the ancestral arrangement of arthropods has remained unchanged for over 400 million years in the lineages leading to the soft ticks and the prostriate ticks.

Measurement of ulnar flow is helpful in predicting ischemia after radial artery harvest.

BACKGROUND: Despite a negative Allen test, some patients develop hand ischemia after radial artery harvesting. The presence of large interosseous collaterals may reduce the sensitivity of Allen test. To evaluate the combination of ulnar flow measurements and the Allen test as an effective screening technique, we performed Doppler ultrasonography during Allen's maneuver. METHODS: The Allen test was used to select candidates for harvesting radial artery from 80 patients undergoing coronary bypass surgery. RESULTS: Of 71 patients with a negative Allen test, one patient developed hand ischemia. This patient was one of six (7.5 %) possessing low ulnar flow levels (less than 40 ml/min/m(2) during compression of the radial artery). This low-flow group had a higher risk for ischemia of the 71 patients with a negative Allen test. The post-operative flow differed greatly from the pre-operative flow in eight patients (11.3 %), which was likely due to large sacrificed interosseous collaterals. CONCLUSION: Combined use of ulnar flow measurement with the Allen test appears to increase the sensitivity of the Allen test. Neither test, however, is sufficient for a group of patients with large interosseous collaterals.

Phylogenic study of denaturation of lactate dehydrogenase isoenzymes from different species by high and low temperature.

We studied the influence of storage at different temperatures on lactate dehydrogenase (EC 1.1.1.27; LD) isoenzymes from different tissues and different species, and analysed biochemical and biophysical mechanisms of denaturation during storage. Isoenzymes obtained from tissue extracts of mammals, poultry, reptiles. amphibians and fish were shown to have their own denaturation ranges at low temperatures by post-treatment assays and transition temperature analysis. These ranges were between -10 and -20 degrees C for most vertebrate LD isoenzymes. Circular dichroism analysis indicated that the denaturation of LD isoenzymes was probably caused by a change in the hydrophobic interactions in the molecule. At higher temperatures, LD-1 isoenzyme was more thermostable than LD-5 from the same animal species, except for rats, the LD-5 activity of which was more thermostable than the LD-1 activity. These findings indicate that variable effects of storage of samples and reference materials at low temperatures should be considered, and that it is necessary to establish LD isoenzyme standards for animal clinical laboratory investigations.

Scope-guided implantation of a stented elephant trunk in acute aortic dissection.

A reliable guide is essential for implanting a stented graft safely into a recently dissected, fragile aorta. In 4 patients with acute aortic dissection, the implantation of a stented elephant trunk was done safely using an endoscope for direct visualization. In all patients, the operation went well. The placement of a stent appears to enhance the benefit of the elephant trunk, which itself reduces the complications of an arch replacement in acute dissection.

Double patch technique for repairing postinfarction ventricular septal defect.

We report 2 cases in which the double patch technique was used to repair an anterior postinfarction ventricular septal defect. To do this, we modified infarct exclusion as follows: In addition to a conventional patch excluding the infarcted muscle, another small patch is used to directly close the septal defect. Gelatin-resorcin-formal glue is applied between the double patches, which prevent the glue from being washed away and enhance it to polymerize stably, thereby rapidly stabilizing the infarcted myocardium with the endocardial patch. Echocardiography immediately after operation showed the infarcted septum had completely adhered to the endocardial patch. Both patients demonstrated satisfactory postoperative hemodynamics. Although 1 patient did well, the other died 6 months postoperatively due to complications of pneumonia and gastrointestinal bleeding secondary to colon carcinoma. This double patch technique appears useful, although further experience is needed to verify its safety and efficacy.

The use of a nondepolarizing cardioplegic solution for cardiac preservation has a beneficial effect on the left ventricular diastolic function.

We have developed a nondepolarizing solution (NDS) that retards myocardial calcium accumulation during cardioplegia. This study compares 1) the membrane resting potential (Em) in Purkinje fibers during cardioplegia induced by NDS or University of Wisconsin solution (UW) at normothermia and hypothermia for 6 h, 2) left ventricular (LV) diastolic function of isolated canine hearts preserved with NDS or UW for 6- and 12 h in hypothermia to elucidate the relationship between diastolic function and myocyte physiology (n = 8, each group), and 3) the effect of Non-depolarizing solution (NDS) compared with Bretschneider's HTK solution on LV diastolic function in isolated rabbit hearts using the Langendorff model in normothermia (n = 10, each group). The membrane resting potential (Em) was as follows: NDS in normothermia, -71 mV (2 min), -65 mV (30 min), and -52 mV (60 min); NDS in hypothermia, -40 mV (1 h) and -32 mV (6 h), while UW in hypothermia 0 mV (6 h). Myocardial calcium accumulation during reperfusion in the NDS groups was minimal and significantly lower than in the UW groups after the 6- and 12 h preservations. Postreperfusion myocardial cyclic adenosin monophosphate (cAMP) and adenosin triphosphate (ATP) concentrations in the NDS groups were closer to normal than in the UW groups after the 6- and 12 h preservations. The postreperfusion myocardial Ca concentration correlated with the cAMP (r = -0.68, n = 25, P = 0.003) and cyclic guanosine monophosphate (cGMP) concentrations (r = -0.69, n = 25, P = 0.003). The left ventricular end-diastolic pressure (LVEDP) after reperfusion correlated with myocardial ATP (r = -0.65, n = 25, P = 0.003) and Ca concentrations (r = -0.68, n = 25, P = 0005). However, the parameter indicating LV elasticity (max LV -dp/dt) correlated with neither the Ca or ATP concentration following reperfusion. NDS prevented stiffness (increased LVEDP) better than HTK during normethermic cardioplegia for 30 min. These results in vitro suggest that NDS prevents myocardial Ca accumulation, depletion of ATP and cAMP, and preserves LV diastolic function, particularly stiffness after reperfusion, for up to 12 h. Furthermore, the myocardial Ca concentration is inversely correlated with the cAMP and cGMP concentrations.

Rotating frame analog of spin-locking and spin-lattice relaxation in the doubly rotating frame.

The present paper reports the achievement of the rotating-frame analog of spin-locking and its application to the precise measurements of the spin-lattice relaxation time T(1DR) in the doubly rotating frame. After the magnetization is aligned along the resonant RF field H(1), a pulse sequence of a low-frequency oscillating magnetic field at exact resonance is applied perpendicular to H(1). We have overcome several technical difficulties arising from the fact that the rotating-wave approximation is not valid for the low-frequency field. We have theoretically derived an expression of T(-1)(1DR) due to fluctuating magnetic dipole interactions in the weak collision case and found an important relation among the spin-lattice relaxation rates T(-1)(1), T(-1)(1rho), and T(-1)(1DR). This relation can be used to ascertain whether the relaxation is only due to the fluctuating magnetic dipole interactions between like spins. The experiment was carried out on (1)H nuclei in tetramethylammonium iodide (CH(3))(4)NI and the temperature dependence of T(-1)(1DR) was measured together with that of T(-1)(1) and T(-1)(1rho). The activation energies and the preexponential factors of Arrhenius expressions of the correlation times are newly determined.

Thickened intima of the aortic arch is a risk factor for stroke with coronary artery bypass grafting.

BACKGROUND: Perioperative stroke is one of the most serious complications of cardiac surgery. METHODS: Using transesophageal echocardiography, we estimated the intimal thickness of the thoracic aorta as an index of the severity of aortic atherosclerosis to determine the risk of stroke in coronary artery bypass grafting (CABG) patients. The study population comprised 315 consecutive patients who underwent isolated CABG with cardiopulmonary bypass. RESULTS: Five patients (1.6%) had perioperative cerebral stroke or systemic emboli. We compared the mean intimal thicknesses of the ascending aorta, aortic arch, and descending aorta. Mean thicknesses in patients without stroke were 2.07 +/- 0.76, 2.78 +/- 1.15, and 2.32 +/- 1.21 mm, respectively, and mean thicknesses in the stroke patients were 1.94 +/- 0.55, 6.94 +/- 3.79, and 3.39 +/- 1.85 mm, respectively. The patients with an intima of more than 5 mm at the aortic arch had a significantly greater incidence of perioperative stroke (p = 0.007). CONCLUSIONS: These results suggest that patients who have an aortic arch intima thickened to more than 5 mm are at a significantly high risk for perioperative stroke, and thus, the CABG procedure should be carefully evaluated to prevent such complications.

Distribution of a major lysosomal membrane glycoprotein, LGP85/LIMP II, in rat tissues.

We previously identified and characterized a major lysosomal membrane glycoprotein, termed LGP85 (identical to LIMP II), in rat liver lysosomes. This study describes the distribution of the mRNA and protein of LGP85 in rat tissues. LGP85 protein and mRNA were detectable in all tissues when analyzed by Western and Northern blotting. The 4.2- and 2.2-kb transcripts of LGP85 were detected in all tissues. Liver and lung have the highest and lowest levels of LGP85 mRNA, respectively. A single protein band with an apparent molecular weight (Mr) of approximately 85000 was detected in each tissue. The specific protein content of LGP85 in spleen was markedly higher than in other tissues. LGP85 protein is distributed in the tissues independently of LGP85 mRNA. Furthermore, there was a less significant relationship between LGP85 protein and another lysosomal membrane glycoprotein, lamp-1, in the tissue distribution (a regression coefficient of 0.086), which suggests that LGP85 may function in vivo independently of lamp-1.

Two acidic amino acid residues, Asp(470) and Glu(471), contained in the carboxyl cytoplasmic tail of a major lysosomal membrane protein, LGP85/LIMP II, are important for its accumulation in secondary lysosomes.

Lysosomal membrane glycoprotein termed LGP85 or LIMP II has a COOH-terminal cytoplasmic tail whose amino acid sequence is R(459)GQGSMDEGTADERAPLIRT(478). Two acidic amino acid residues, D(470) and E(471), in the cytoplasmic tail of LGP85 are crucial for its binding to adaptor-like complex AP-3. In the present study we investigated their role(s) in intracellular distributions of LGP85 using two alanine substitution mutants at D(470) and E(471) (defined as D470A and E471A, respectively). Immunofluorescence analysis showed that D470A and E471A are localized to endocytic organelles as well as wild-type LGP85. However, the subcellular fractionation study revealed that D470A and E471A are different from wild-type LGP85 in the distribution among early endosomes, late endosomes, and lysosomes. A major portion of wild-type LGP85 existed in the densest lysosomal fraction. In contrast, a significant amount of D470A existed in the early endosomal fraction with a light buoyant density, while less D470A resided in the lysosomal fraction. E471A broadened from the early endosomal fraction to the lysosomal fraction without the high lysosomal peak. These findings indicate that the two acidic residues, D(470) and E(471), play an important role in regulation of LGP85 movement within the endocytic pathway, which finally makes the highest concentration of LGP85 in the dense secondary lysosomes.

Acylzirconocene Chloride as an "Unmasked" Acyl Anion: Enantioselective 1,2-Addition to alpha,beta-Unsaturated Ketone Derivatives.

The chiral monodentate phosphane ligand (R)-MOP facilitated the first enantioselective nucleophilic 1,2-addition of an "unmasked" acyl anion to a carbonyl group in the Pd(II)-catalyzed reaction of alpha,beta-unsaturated ketones with acylzirconocene chlorides [Eq. (1), 66 % ee, 88 % yield; (R)-MOP=(R)-2-(diphenylphosphanyl)-2'-methoxy-1,1'-binaphthyl].

Identification and characterization of a major lysosomal membrane glycoprotein, LGP85/LIMP II in mouse liver.

We previously have purified and characterized a major lysosomal membrane glycoprotein termed LGP85 (LIMP II) in rat liver lysosomes. In this study, LGP85 in mouse liver lysosomes was identified and characterized by biochemical and molecular biological methods. Lysosomal membranes were isolated from murine liver by differential centrifugation. LGP85 was present in the lysosomal membrane fraction from mouse liver in a comparable amount to another lysosomal membrane glycoprotein, lamp-2. Mouse LGP85 (M-LGP85) from liver lysosomal membranes exhibited an Mr of 80,000 on SDS-PAGE, which is smaller by 5,000 than that of rat LGP85 (R-LGP85). M-LGP85 was immunochemically detected in the extracts of brain, heart, lung, liver, and kidney. A cDNA encoding M-LGP85 was cloned from mouse liver cDNA library. The primary protein structure deduced from a nucleotide sequence of M-LGP85 cDNA indicated that M-LGP85 consists of 478 amino acids with Mr of 54,069. M-LGP85 showed 93.3 and 86.0% sequence similarities to its rat and human counterparts in amino acids, respectively. M-LGP85 contains 11 potential N-glycosylation sites which are heavily glycosylated, resulting in the increased Mr of M-LGP85 present in the mouse liver lysosomes. It is likely that M-LGP85 traverses the lysosomal membrane twice, with an NH2-terminal transmembrane domain, and another hydrophobic domain near the COOH-terminus. M-LGP85 has a protruding COOH-terminal cytoplasmic tail consisting of amino acid residues including the leucine-isoleucine sequence shown to be the lysosomal targeting signal of R-LGP85 and human LGP85 (H-LGP85). The high level of expression of M-LGP85 in the lysosomal membrane, the high structural similarities among M-, R-, and H-LGP85, and the occurrence of M-LGP85 in all the mouse tissues examined suggest the essential and constitutive function of LGP85 in lysosomes.

Gelatin use impairs platelet adhesion during cardiac surgery.

Artificial colloids based on gelatin are used as plasma expander to replace donor blood products. In laboratory experiments, gelatin reduced both the velocity and extend of platelet agglutination by ristocetin, and only the agglutination velocity by polybrene (p < 0.05). Furthermore, gelatin delayed the in-vitro platelet plug formation under shear-stress in the absence of ADP (p < 0.05), whereas gelatin induced no delay in the presence of ADP. Thus, after induction of vWF release from platelets by polybrene or ADP, platelet function was normal. These results indicate that gelatin affects in particular the functionality of plasma-vWF and partly inhibits platelet adhesion. These negative effects of gelatin on hemostasis were demonstrated in two clinical studies during cardiac surgery. In a randomized study of sixty patients undergoing cardiac surgery, gelatin as prime in the heart-lung machine appeared to result in diminished efficacy of aprotinin on hemostasis, whereas it did not affect hemostasis in non-aprotinin patients. An additional retrospective clinical study showed that only high dose of gelatin affected hemostasis. This suggests a limited role of plasma-vWF and a strong back-up mechanism of platelet-vWF in achieving hemostasis.

Hemostatic function of aspirin-treated platelets vulnerable to cardiopulmonary bypass. Altered shear-induced pathway.

The impaired hemostasis of aspirin-treated patients is an annoying problem during and after cardiopulmonary bypass. The hemostatic function of platelets comprises two mechanisms: the shear-induced and the cyclooxygenase pathways. Because the latter is inhibited in aspirin-treated patients, the hemostatic function depends mainly on the former pathway. To investigate the effect of cardiopulmonary bypass on the shear-induced pathway, a double-blind study of preoperative aspirin treatment (325 mg) and placebo was conducted in 40 patients undergoing coronary artery bypass grafting. Postoperative blood loss was higher in the aspirin-treated patients than in the placebo-treated patients (p < 0.05). The shear-induced hemostasis was monitored by the in vitro bleeding test (Thrombostat), which mimics bleeding through an injured arteriole. The shear-induced pathway of aspirin-treated platelets was not affected before cardiopulmonary bypass, but it was impaired more during the operation (p < 0.01) and remained worse afterward (p < 0.05), compared with that of placebo-treated platelets. The inhibitory effects of aspirin on thromboxane production and on collagen-induced platelet aggregation remained throughout the operation. In aspirin-treated platelets, the aggregation capacity induced by adenosine diphosphate was inhibited before the operation (p < 0.05) and showed substantial recovery during the operation (p < 0.05). These results suggest that the shear-induced pathway of aspirin-treated platelets is more vulnerable to cardiopulmonary bypass than the pathway in normal platelets and causes severe impairment of hemostasis afterward.