Importance of a biofouling-resistant phospholipid polymer to create a heparinized blood-compatible surface

Heparinization is believed to be one of the methods to suppress thrombus formation on blood-contacting surfaces. However, this study hypothesizes that heparinization alone might not be sufficient to provide a blood-compatible surface; that is, a surface property that resists biofouling is necessary to obtain an effective heparin-modified surface. 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers with 2-aminoethyl methacrylate (AEMA) were synthesized to immobilize heparin through ionic bonding. The primary amino groups of AEMA were considered to be the polymer surface because the zeta-potential of the surface was positive when the mole fraction of the AEMA units was above 0.2. The antithrombogenic character of the polymer surface modified with heparin was evaluated by both Lee-White and microsphere column methods. The coagulation period of human whole blood in the absence of anticoagulant in glass tubing coated with the MPC polymer was longer than that in the original glass tube. Cell adhesion was completely inhibited on the MPC polymer surface after contact with human whole blood without anticoagulant. However, many adherent blood cells were observed on poly(2-ethylhexyl methacrylate-co-AEMA) (no MPC unit) even after heparinization. These results strongly indicate that the MPC polymer is a useful substrate where the heparin works well and that the heparin-immobilized MPC polymer has superior blood compatibility to the simple MPC polymer.     

Increase of soluble Fc gamma RIIIa derived from macrophages in plasma from patients with atherosclerosis

Macrophages play a major role in the development of vascular lesions in atherogenesis. The cells express Fc gamma RIIIa(CD16) identical to that in NK cells, but with a cell type-specific glycosylation. In contrast, neutrophils express Fc gamma RIIIb in two allotypes, NA1- and NA2- Fc gamma RIIIb. These Fc gamma RIIIs are released from the cell surface on activation, and these soluble forms(sFc gamma RIII) are present in plasma. In the present study, we measured sFc gamma RIIIaM phi in plasma with Immuno-PCR with newly-developed anti-Fc gamma RIII mAb, MKGR14(mIgM), which recognizes Fc gamma RIIIaM phi specifically. In healthy donors, the level of sFc gamma RIIIaM phi increased with age. In contrast, the sFc gamma RIIIa level correlated with the number of NK cells in peripheral blood, and the level of total sFc gamma RIII(sFc gamma RIIIa plus sFc gamma RIIIb) correlated with the number of neutrophils. There was no correlation among the levels of three sFc gamma RIIIs was observed in healthy donors. The levels of these sFc gamma RIII were significantly increased in patients with coronary artery disease (CAD) compared with age-matched healthy donors. The sFc gamma RIIIaM phi level was related to the number of significant coronary artery stenoses, and correlated with the sFc gamma RIIIa level, total cholesterol, HDL-cholesterol(negatively), LDL to HDL ratio, triglycerides and body mass index. These findings may show that the macrophages are activated during the incipient stage of atherosclerosis, and that sFc gamma RIIIaM phi may serve as predictive marker for atherosclerosis.     

Mcm1 regulates donor preference controlled by the recombination enhancer in Saccharomyces mating-type switching

Switching of Saccharomyces mating type by replacement of sequences at the MAT locus involves a choice between two donors, HML and HMR. MATα cells inhibit recombination along the entire left arm of chromosome III, including HML, whereas MATa cells activate this same region. MATa-dependent activation of HML depends on a small, cis-acting DNA sequence designated the recombination enhancer (RE), located 17 kb centromere-proximal to HML. A comparison of RE sequences interchangeable between Saccharomyces cerevisiae and Saccharomyces carlsbergensis defines a minimum RE of 244 bp. RE activity is repressed in MATα cells by binding of the Matα2–Mcm1 corepressor to a site within the RE. Mutation of the two Matα2 binding sites removes most, but not all, of this repression, and RE chromatin structure in MATα cells becomes indistinguishable from that seen in MATa. Surprisingly, a 2-bp mutation in the Mcm1 binding site completely abolishes RE activity in MATa cells; moreover, RE chromatin structure in the MATa mutant becomes very similar to that seen in MATα cells with a normal RE, displaying highly ordered nucleosomes despite the absence of Matα2. Further, a mutation that alters the ability of Mcm1 to act with Matα2 in repressing a-specific genes also alters donor preference in either mating type. Thus, Mcm1 is critically responsible for the activation as well as the Matα2-Mcm1-mediated repression of RE activity.     

Antioxidant enzyme systems in skeletal muscle atrophied by immobilization

To clarify the mechanism of oxidative stress in skeletal muscle atrophied by immobilization, we investigated the change of antioxidant enzyme activities in a typical slow red muscle, the soleus. Atrophied soleus muscles were collected from male Wistar rats (16 weeks old), one ankle joint of which had been immobilized in the fully extended position for 7 days. Also, soleus muscles were collected from intact age-matched rats as control. The activities of Mn-containing superoxide dismutase (Mn-SOD), Cu,Zn-containing superoxide dismutase (Cu,Zn-SOD), Se-dependent glutathione peroxidase (Se-GSHPx), glutathione S-transferase (GST), catalase, and glutathione reductase (GSSGRx) were measured. The activities of Cu,Zn-SOD, GST, and GSSGRx were significantly higher in atrophied muscles, while the others were unchanged. Increased Cu,Zn-SOD and unchanged Mn-SOD levels might reflect increased generation of superoxide anions in the cytoplasm rather than in the mitochondria. Owing to the enhancement of Cu,Zn-SOD and the unaltered Se-GSHPx and catalase activities, hydrogen peroxide is thought to be increased in the cytoplasm. Because there is also an increase of iron in the microsomes of atrophied muscles, the production of hydroxyl radicals, the most aggressive of radicals, might consequently be elevated.     

Polymorphisms and the differential antiviral activity of the chicken Mx gene

The nucleotide sequence of chicken Mx cDNA was reported earlier using the White Leghorn breed in Germany, but it showed no enhanced resistance to viruses. In this study, the nucleotide sequences of chicken Mx cDNA were determined in many breeds. A total of 25 nucleotide substitutions, of which 14 were deduced to cause amino acid exchanges, were detected, suggesting that the chicken Mx gene is very polymorphic. Transfected cell clones expressing chicken Mx mRNA were established after the Mx cDNA was constructed with an expression vector and introduced into mouse 3T3 cells, and the Mx genes from some breeds were demonstrated to confer positive antiviral responses to influenza virus and vesicular stomatitis virus. On the basis of the comparison among the antiviral activities associated with many Mx variations, a specific amino acid substitution at position 631 (Ser to Asn) was considered to determine the antivirally positive or negative Mx gene. Thus, a single amino acid substitution influences the antiviral activity of Mx in domesticated chickens.     

Evaluation of endocrine disrupting activity of plasticizers in polyvinyl chloride tubes by estrogen receptor alpha binding assay

Polyvinyl chloride (PVC) tubing is an indispensable medical material for extracorporeal circulation therapy. However, di(2-ethylhexyl)phthalate (DEHP), a suspected endocrine disruptor, can be eluted from PVC, suggesting that an alternative material that does not contain DEHP is needed for clinical applications. First, we evaluated the endocrine disrupting risks of the plasticizers contained in PVC tubes by investigating their binding affinities for the human estrogen receptor alpha (ERα). Our results revealed that, while DEHP has some binding affinity for ERα, neither epoxidized soybean oil nor tris(2-ethylhexyl)trimellitate (an alternative to DEHP) has any affinity for ERα. Second, we evaluated the endocrine disrupting risks of a tube made of newly developed plasticizer-free (PF) materials. We confirmed the presence of DEHP and detected several unidentified substances in plasma stored within the PVC tube. This plasma's competitive binding affinity for ERα was significantly higher than that of control plasma (P < 0.01). In contrast, the profile of plasma stored in the PF tube was similar to that of the control, both in terms of high-performance liquid chromatography chromatograms and competitive binding capacity for ERα, suggesting that the PF tube is biocompatible and is useful for reducing the elution of substances capable of binding to ERα. Presented in part at the 42nd Congress of the Japanese Society for Artificial Organs, October 5–7, 2004, Tokyo, Japan     

Morphological study of disordered myelination and the degeneration of nerve fibers in the spinal cord of mice lacking complex gangliosides

Gangliosides, a family of glycosphingolipids that contain sialic acid, are abundant on the neuronal cell membranes, but their precise functions in the central nervous system remain largely undefined. In a previous study of GalNAc-T(-/-) mice engineered to lack beta1,4-N-acetylgalactos-aminyltransferase (GM2/GD2 synthase) to abolish any, complex gangliosides, we observed the reduction of nerve conduction velocity but did not find any obvious morphological change in the brain. In the present study, we observed morphological changes in the nerve fiber tracts of the spinal cord in these mice. In GalNAc-T(-/-) mice, the number of degenerated axons was markedly increased in the dorsal funiculus, tract of Lissauer, and dorsolateral funiculus of the cervical segment of the spinal cord as well as the dorsal funiculus and tract of Lissauer of the lumbar segment of the spinal cord. There were also increased numbers of unmyelinated fibers in GalNAc-T(-/-) mice. Loosened myelin sheaths and myelin sheaths separated from axons by wide spaces were also observed in GalNAc-T(-/-) mice. These results provide a morphological basis for the previously observed reduction in the nerve conduction velocity and suggest that complex gangliosides are essential for the maintenance of myelin and the integrity of nerve fibers of the spinal cord.     

Quantitative evaluation of hand cranking a roller pump in a crisis management drill

The heart-lung machines for open-heart surgery have improved over the past 50 years; they rarely break down and are almost always equipped with backup batteries. The hand-cranking procedure only becomes necessary when a pump breaks down during perfusion or after the batteries have run out. In this study, the performance of hand cranking a roller pump was quantitatively assessed by an objective method using the ECCSIM-Lite educational simulator system. A roller pump connected to an extracorporeal circuit with an oxygenator and with gravity venous drainage was used. A flow sensor unit consisting of electromagnetic sensors was used to measure arterial and venous flow rates, and a built-in pressure sensor was used to measure the water level in the reservoir. A preliminary study of continuous cranking by a team of six people was conducted as a surprise drill. This system was then used at a perfusion seminar. At the seminar, 1-min hand-cranking drills were conducted by volunteers according to a prepared scenario. The data were calculated on site and trend graphs of individual performances were given to the participants as a handout. Preliminary studies showed that each person's performance was different. Results from 1-min drills showed that good performance was not related to the number of clinical cases experienced, years of practice, or experience in hand cranking. Hand cranking to maintain the target flow rate could be achieved without practice; however, manipulating the venous return clamp requires practice. While the necessity of performing hand cranking during perfusion due to pump failure is rare, we believe that it is beneficial for perfusionists and patients to include hand-cranking practice in periodic extracorporeal circulation crisis management drills because a drill allows perfusionists to mentally rehearse the procedures should such a crisis occur.