Hiroyuki Kuma

Crystal structure of the anion exchanger domain of human erythrocyte band 3.

Getting rid of carbon dioxide In mammals, red blood cells deliver oxygen to tissues and remove carbon dioxide. Key to this essential process is a membrane protein called anion exchanger 1 (AE1) which transports bicarbonate (formed from carbon dioxide) out of red blood cells in exchange for chloride. This decreases the pH inside the blood cells, so that oxygen is released from hemoglobin and can diffuse into tissues. Arakawa et al. report the crystal structure of the transmembrane anion exchanger domain of AE1, which includes 14 transmembrane helices. The structure provides a basis for understanding the effects of mutations that lead to red blood cell diseases and also gives insight into the mechanism of ion transport. Science, this issue p. 680 The structure of a key red blood cell membrane protein provides a basis for understanding mutations that lead to red blood cell diseases. Anion exchanger 1 (AE1), also known as band 3 or SLC4A1, plays a key role in the removal of carbon dioxide from tissues by facilitating the exchange of chloride and bicarbonate across the plasma membrane of erythrocytes. An isoform of AE1 is also present in the kidney. Specific mutations in human AE1 cause several types of hereditary hemolytic anemias and/or distal renal tubular acidosis. Here we report the crystal structure of the band 3 anion exchanger domain (AE1CTD) at 3.5 angstroms. The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed us to identify the anion-binding position in the AE1CTD, and to propose a possible transport mechanism that could explain why selected mutations lead to disease.

Structure of the Membrane Domain of Human Erythrocyte Anion Exchanger 1 Revealed by Electron Crystallography

The membrane domain of human erythrocyte anion exchanger 1 (AE1) works as a Cl(-)/HCO(3)(-) antiporter. This exchange is a key step for CO(2)/O(2) circulation in the blood. In spite of their importance, structural information about AE1 and the AE (anion exchanger) family are still very limited. We used electron microscopy to solve the three-dimensional structure of the AE1 membrane domain, fixed in an outward-open conformation by cross-linking, at 7.5-A resolution. A dimer of AE1 membrane domains packed in two-dimensional array showed a projection map similar to that of the prokaryotic homolog of the ClC chloride channel, a Cl(-)/H(+) antiporter. In a three-dimensional map, there are V-shaped densities near the center of the dimer and slightly narrower V-shaped clusters at a greater distance from the center of the dimer. These appear to be inserted into the membrane from opposite sides. The structural motifs, two homologous pairs of helices in internal repeats of the ClC transporter (helices B+C and J+K), are well fitted to those AE1 densities after simple domain movement.

Fast Detection of Biological Targets With Magnetic Marker and SQUID

We have been developing a SQUID system for the detection of biological targets. In this system, magnetic markers are bound to the targets, and the magnetic signal from the bound markers is detected with the SQUID. In order to realize fast detection of the targets, we developed a liquid-phase detection method. First, we used large polymer beads as material to capture the targets. Since the polymer beads are uniformly dispersed in liquid, biological targets on the surface of the polymer bead can be easily coupled to the markers, which results in the fast reaction time. Next, we detected the bound markers without using the washing process to separate the bound and unbound markers, which was realized by using the difference in the Brownian relaxation time between them. Using this procedure, we demonstrated the detection of the target called IgE, as well as biotin-coated polymer beads. We obtained a good relationship between the amount of IgE and the magnetic signal. The result was the same as that obtained using the conventional procedure. The reaction time for the coupling between the magnetic marker and the target was 4 min, which was much shorter than the conventional method. These results show the usefulness of the present method.

Liquid Phase Immunoassay Using Magnetic Markers and Superconducting Quantum Interference Device

A liquid phase immunoassay utilizing magnetic markers and a high-Tc superconducting quantum interference device (SQUID) was studied. In this method, the biological target is detected using magnetic markers, i.e., the magnetic signal from the markers that bound to the target is detected with the SQUID. The detection was performed in a solution containing both the bound and unbound (free) markers without using the so-called bound/free (BF) separation process. The bound markers were distinguished from the free markers by utilizing the Brownian rotation of the free markers. First, the properties of the free markers in the solution, such as the M–H curve and magnetic relaxation, were measured to study the background signal from the free markers. Markers that exhibit remanence were used for the experiment. Using the obtained results, we discuss the effects of the residual earth field and aggregation of the markers on the background signal. Next, we detected a fungus, Candida albicans, with the described liquid phase immunoassay. Good relationship was obtained between the detected signal and the number of fungi. The minimum detectable number of fungi was as small as 30.

Characterization of magnetic markers for liquid-phase immunoassays using brownian relaxation

We characterized the magnetic markers used in biological immunoassays based on Brownian relaxation. Because the markers are composed of aggregated nanoparticles, i.e., magnetic nanoclusters, we first clarified their magnetic properties using AC susceptibility measurements, magnetization (M–H) curves, and magnetic relaxation properties. Analyzing the experimental results, we obtained the key parameters for the immunoassay, i.e., hydrodynamic diameter dh, magnetic moment mB, and anisotropy energy EB of the markers. Because these parameters were distributed in practical samples, we took their distribution into account in the analysis. Next, we showed the relationship between these parameters obtained from different samples. It was shown that mB increased approximately in proportion to dh. On the other hand, no clear correlation between mB and EB was obtained. These results were very different from those expected from single-domain nanoparticles and must be taken into account when magnetic markers are used in immunoassays based on Brownian relaxation.

Liquid phase immunoassays utilizing magnetic markers and SQUID magnetometer.

Abstract Background: Immunoassays are one main detection system used in the field of clinical chemistry. Recent developments of a new detection method utilizing a magnetic marker and magnetic sensor have enabled rapid and sensitive immunoassay without the need for bound/free (BF) separation. Methods: Newly-synthesized conjugated avidin was used as the magnetic marker for quantitative analysis of human interleukin-8 (hIL-8) and immunoglobulin E (hIgE) in several media. A superconducting quantum interference device sensor detected the magnetic fields from markers fixed to antigens by the sandwich method. Magnetic signals from unbound markers were nearly zero due to Brownian rotation. Results: Our magnetic immunoassay could detect four attomoles of model proteins (hIL-8, hIgE) in phosphate buffer without BF separation. Using our standard curve, the range of protein detected ranged from 40 femtomoles to 4 attomoles, and we observed a strong association between protein amounts and magnetic signals from the bound markers. The homogeneous immunoassay could also quantify three hundred cells from the fungus Candida albicans in phosphate buffer. Conclusions: The present study demonstrates the ability of magnetic markers for measuring biological targets without BF separation. This detection system has great potential for use as the next generations analytical system. Clin Chem Lab Med 2010;48:1263–9.

Development of a SQUID System Using Field Reversal for Rapidly Detecting Bacteria

Pathogen identification usually requires growth of the pathogen by culture, which requires considerable time and manipulation by an experienced operator, leading to delays in diagnosis and treatment. We have investigated pathogen detection using a highly sensitive HTS-SQUID and magnetic markers and have developed a rapid and simple pathogen detection method. The magnetic markers, magnetic nanoparticles coated with detecting antibodies, bind to the target substance (antigen). The magnetic signal of the bound markers is measured with the highly sensitive SQUID. A remarkable feature of the magnetic assay is the disappearance of the magnetic signal from the unbound markers due to Brownian rotation. This makes it possible to detect the magnetic signal of the bound magnetic markers without removal of the unbound markers. In practice, however, the residual field around the SQUID generates an undesired magnetic signal from the unbound markers as a result of biased Brownian rotation. We developed a field reversal method - a measurement scheme - that eliminates the magnetic signal from the unbound markers. A difference signal is obtained by subtracting the magnetic signals measured by applying a magnetization field in two directions. The validity of this method was demonstrated experimentally using polymer beads as simulated bacteria. Its feasibility was demonstrated by the detection of Candida albicans, a pathogenic fungus. A magnetic signal of 3 mPhi0 was detected from a sample containing 300 cells of Candida albicans. The detection limit was estimated from the system noise level of 0.5 mPhi0 to be about one hundred cells of Candida albicans, indicating that this method has high sensitivity. These results show that magnetic assay using a highly sensitive SQUID can provide rapid and simple pathogen testing without culture.

Beneficial effect of EPA (20:5 n-3 PUFA) on preventing venous thromboembolism: a rat tail thrombosis model experiment.

Recent randomized controlled trials on coronary heart disease failed to demonstrate benefits of long chain n-3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) [1,2], although EPA and DHA have been recognized to decrease the risk of cardiovascular disease [3]. Herein, however, using a rat tail thrombosis model, we showed a new beneficial effect of EPA in preventing venous thromboembolism. Individuals prone to developing thrombosis have thrombophilia. Among Caucasians, “Activated protein C (APC)-resistance” is the major thrombophilia [4] and “APC-dysfunction” is the major thrombophilia in Japanese and Chinese populations [5]. These thrombophilias both tend to promote thrombosis due to the relatively weak APC anticoagulation reaction to blood coagulation [5,6]. We have examined the effects of phospholipids on blood coagulation and APC anticoagulation reactions, and found phospholipids of phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and phosphatidylethanolamine (PE) to exert complicated effects on the coagulation and anticoagulation reactions. That is, PC strongly and dose-dependently inhibited APC and protein S activities and the FXa-prothrombin reaction but once PC was converted to LPC by lipase, LPC accelerated the FXa-prothrombin reaction [7,8]. Protein S activity was measured as the enhancement in the rate of APC activity in response to adding protein S to the APC-FVa reaction

Genotyping analysis of protein S-Tokushima (K196E) and the involvement of protein S antigen and activity in patients with recurrent pregnancy loss

OBJECTIVE Preston et al. indicated that Protein S (PS) deficiency was associated with stillbirths but not miscarriages. The PS-Tokushima missense variant was reported to serve as a genetic risk factor for deep vein thrombosis in the Japanese population. A previous cross-sectional study showed no increase in the prevalence of PS-Tokushima in patients with recurrent early pregnancy loss or in patients with intra uterine fetal death and/or fetal growth restriction. There has been limited number of prospective studies examining the pregnancy outcome in patients with both a PS deficiency and recurrent pregnancy loss (RPL). We examined the association between PS deficiency, PS-Tokushima and RPL. STUDY DESIGN The study group consisted of 355 Japanese women with two or more consecutive pregnancy losses and 101 parous women. The frequency of PS-Tokushima and the subsequent live birth rate in relation to a PS deficiency defined as low PS-specific activity (total PS activity/total PS antigen) and the carriage of PS-Tokushima were examined. RESULTS AND CONCLUSIONS There was no significant difference in the frequency of PS-Tokushima between patients and controls. The 8 patients carriers of PS-Tokushima variant were capable of a subsequent live birth without the use of heparin. There was no significant difference in subsequent live birth rates between patients with low or normal PS-specific activity/PS activity without heparin prophylaxis after excluding miscarriages caused by an abnormal embryonic karyotype using multivariate logistic regression analysis. There was no association between PS-Tokushima and RPL and a PS deficiency or low PS activity was shown not to serve as a reliable clinical predictor of subsequent miscarriage.