Large Diamagnetism and Electromagnetic Duality in Two-Dimensional Dirac Electron System.

A Dirac electron system in solids mimics relativistic quantum physics that is compatible with Maxwell's equations, with which we anticipate unified electromagnetic responses. We find a large orbital diamagnetism only along the interplane direction and a nearly temperature-independent electrical conductivity of the order of e^{2}/h per plane for the new 2D Dirac organic conductor, α-(BETS)_{2}I_{3}, where BETS is bis(ethylenedithio)tetraselenafulvalene. Unlike conventional electrons in solids whose nonrelativistic effects bifurcate electric and magnetic responses, the observed orbital diamagnetism scales with the electrical conductivity in a wide temperature range. This demonstrates that an electromagnetic duality that is valid only within the relativistic framework is revived in solids.

Rapid detection of ciguatoxins in Gambierdiscus and Fukuyoa with immunosensing tools.

Consumption of seafood contaminated with ciguatoxins (CTXs) leads to a foodborne disease known as ciguatera. Primary producers of CTXs are epibenthic dinoflagellates of the genera Gambierdiscus and Fukuyoa. In this study, thirteen Gambierdiscus and Fukuyoa strains were cultured, harvested at exponential phase, and CTXs were extracted with an implemented rapid protocol. Microalgal extracts were obtained from pellets with a low cell abundance (20,000 cell/mL) and were then analyzed with magnetic bead (MB)-based immunosensing tools (colorimetric immunoassay and electrochemical immunosensor). It is the first time that these approaches are used to screen Gambierdiscus and Fukuyoa strains, providing not only a global indication of the presence of CTXs, but also the ability to discriminate between two series of congeners (CTX1B and CTX3C). Analysis of the microalgal extracts revealed the presence of CTXs in 11 out of 13 strains and provided new information about Gambierdiscus and Fukuyoa toxin profiles. The use of immunosensing tools in the analysis of microalgal extracts facilitates the elucidation of further knowledge regarding these dinoflagellate genera and can contribute to improved ciguatera risk assessment and management.

Energy consumption in a baffled membrane bioreactor (B-MBR): estimation based on long-term continuous operation.

We investigated the operating conditions of a baffled membrane bioreactor (B-MBR) under which long-term stable operation can be achieved through the continuous operation of a pilot-scale B-MBR. Under appropriate operating conditions, the B-MBR was capable of achieving excellent treated water quality in terms of biochemical oxygen demand and concentration of total nitrogen. Excellent removal of total phosphorus was also achieved. In addition, the degree of membrane fouling was acceptable, indicating that stable continuous operation of a B-MBR is possible under the operating conditions adopted in the present study. Estimation of the specific energy consumption in hypothetical full-scale B-MBRs operated under the conditions recommended by the findings was also performed in this study. The results suggest that energy consumption in full-scale B-MBRs would be in the range of 0.20-0.22 kWh/m3. These results strongly suggest that energy consumption in MBR operation can be significantly reduced by applying the concept of a B-MBR.

Effects of recirculation and separation times on nitrogen removal in baffled membrane bioreactor (B-MBR).

In this study, we investigated the effects of recirculation and separation times on removals of organic matter, nitrogen, and phosphorus in a baffled membrane bioreactor (B-MBR) treating real municipal wastewater. A pilot-scale B-MBR experimental apparatus was operated under two different sets of recirculation and separation times. The results revealed that, irrespective of operating conditions, the biochemical oxygen demand (BOD) and concentration of total nitrogen (T-N) in the treated water can be lowered to less than 3 and 5 mg/L, respectively. Although T-N was effectively removed in the two different operating conditions, increase in the fraction of recirculation time results in tiny deterioration of nitrogen removal efficiency in the B-MBR. Phosphorus removal efficiency was also slightly decreased as the fraction of recirculation time (ratio between recirculation and separation times) was increased. The results of the measurement of dissolved oxygen (DO) profiles at different points of the B-MBR apparatus indicate that the increase in DO concentration in the anoxic zone of the B-MBR becomes much more pronounced by increasing recirculation intensity. On the basis of the results obtained in this study, it can be concluded that efficient removal of BOD, T-N, and total phosphorus can be achieved by the B-MBR as long as appropriate recirculation intensity is selected.

Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface.

A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of alpha-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.

Catalytic antibodies induced by a zwitterionic hapten.

A zwitterionic hapten 4 featuring both positively and negatively charged functional groups was designed and synthesized with the goal of generating catalytic antibodies for the hydrolysis of ester 6 and amide 7. Of the 36 monoclonal antibodies specific to BSA-4 (bovine serum albumin) that were isolated, six accelerated the hydrolysis of 6. Two catalytic antibodies with distinctively different and representative kinetic behaviors were selected for detailed kinetic studies. Whereas H8-2-6F11 showed burst kinetic behavior, which can be attributed to the formation of an acyl intermediate, H8-1-2D5 did not, but it did exhibit high multiple turnover activity. The rate of hydrolysis of 6 catalyzed by H8-1-2D5 followed Michaelis-Menten kinetics; the apparent values of the Michaelis-Menten constant Km and the catalytic constant kcat were 488 microM and 3.5 min(-1), respectively. The catalytic rate enhancement (kcat/kun) observed for H8-1-2D5 was 1.3 x 10(5), which is approximately two orders of magnitude greater than those for monofunctional haptens. Thus H8-1-2D5 compares well in catalytic activity with antibodies isolated by a related approach called heterologous immunization.

Concise synthesis of ciguatoxin ABC-ring fragments and surface plasmon resonance study of the interaction of their BSA conjugates with monoclonal antibodies.

Monoclonal antibodies (mAbs), 4H2 and 6H7, were prepared previously using a protein conjugate of a 1:1 epimeric mixture of the synthetic ABC-ring fragments of ciguatoxin (CTX), 3 and 4. Here, the interactions of these mAbs with the fragments of CTX and CTX3C, 3 and 5, were investigated by surface plasmon resonance (SPR) spectroscopy in an attempt to clarify an antigenic determinant. Compared with the previous synthesis, the fragment 3 possessing the 2S configuration was synthesized from tri-O-acetyl-D-glucal much more effectively. The mAb 4H2 was already known to show a dose-dependent binding to the bovine serum albumin (BSA) conjugate of 3, but not to that of 5. The present SPR study of 4H2 demonstrates that the A-ring side chain of 3 plays a decisive role as an epitope. Therefore, SPR can effectively replace the ELISA method for the analysis of mAbs.

Antibody-catalyzed removal of the p-nitrobenzyl ester protecting group: the molecular basis of broad substrate specificity.

Antibody catalysts for the removal of the p-nitrobenzyl ester protecting group have been generated to accommodate a broad range of substrates. Antibody 7B9, which was elicited against p-nitrobenzyl phosphonate 1, catalyzed the hydrolyses of p-nitrobenzyl monoesters of nonsubstituted, and beta- and gamma-substituted glutaric acids with almost identical Km and kcat values. In addition, 7B9 displayed substrate tolerance towards the a-substituents and accepted the p-nitrobenzyl esters of Leu, Norleu, and Phe. To define the molecular basis of the broad substrate tolerance, we have cloned and sequenced the antibody and constructed a model of the active-site-hapten complex. The model showed a relatively shallow pocket of the antigen-combining site that accommodates the p-nitrobenzyl moiety, and this is consistent with the observed substrate specificity. Thus, in the antibody-catalyzed reaction, the alpha-, beta-, and gamma-substituents of the substrates should be outside the combining site and ignored by the antibody recognition. A structural comparison of 7B9 with antibody D2.3, elicited against the structurally similar haptenic phosphonate, suggests the significance of the linker moiety in hapten design, which endows antibody catalysts with broad substrate specificity. These investigations provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates for practical applications in organic synthetic chemistry.

Diverse structural solutions to catalysis in a family of antibodies.

BACKGROUND: Small organic molecules coupled to a carrier protein elicit an antibody response on immunisation. The diversity of this response has been found to be very narrow in several cases. Some antibodies also catalyse chemical reactions. Such catalytic antibodies are usually identified among those that bind tightly to an analogue of the transition state (TSA) of the relevant reaction; therefore, catalytic antibodies are also thought to have restricted diversity. To further characterise this diversity, we investigated the structure and biochemistry of the catalytic antibody 7C8, one of the most efficient of those which enhance the hydrolysis of chloramphenicol esters, and compared it to the other catalytic antibodies elicited in the same immunisation. RESULTS: The structure of a complex of the 7C8 antibody Fab fragment with the hapten TSA used to elicit it was determined at 2.2 A resolution. Structural comparison with another catalytic antibody (6D9) raised against the same hapten revealed that the two antibodies use different binding modes. Furthermore, whereas 6D9 catalyses hydrolysis solely by transition-state stabilisation, data on 7C8 show that the two antibodies use mechanisms where the catalytic residue, substrate specificity and rate-limiting step differ. CONCLUSIONS: Our results demonstrate that substantial diversity may be present among antibodies catalysing the same reaction. Therefore, some of these antibodies represent different starting points for mutagenesis aimed at boosting their activity. This increases the chance of obtaining more proficient catalysts and provides opportunities for tailoring catalysts with different specificities.

Structure-based design of diaminopyranosides as a novel inhibitor core unit of HIV proteases.

Novel HIV PR inhibitors, which contain a diaminopyranoside moiety as an inhibitor core unit, were designed based on the 3D structures of complexes of HIV-1 PR with transition-state mimics. These compounds were examined for their ability to inhibit the hydrolytic activity of a recombinant HIV-1 PR.

[Immunological mechanism of pregnancy and miscarriage--a study with a murine spontaneous miscarriage model].

In order to investigate the immunological mechanisms of pregnancy, fluorocytometric and immunohistochemical analysis of the cells was performed in the placenta and spleen of a murine spontaneous miscarriage model (CBA/J x DBA/2) and control (CBA/J x BALB/c). There was a significant difference between the miscarriage rate for the miscarriage model and that for the control, even though H-2 in these two group is matched. The analysis also was performed in a miscarriage model immunized with male splenocytes. Moreover, the effect of gamma-interferon, a potentiator of NK cell activity, on pregnancy was examined. Interferon treatment increased the miscarriage rate. In pregnancy, the number of splenocyte positive Asialo-GM1 or LFA-1 decreased and the intensity of these antigens decreased, as well. Interleukin-2R positive cell increased in number as well as intensity. In the miscarriage model group successfully treated by immunization, the number of Asialo-GM1 positive cells and L3T4 positive cells decreased, whereas they increased in the unsuccessfully treated group. Asialo-GM1 positive cells in the placenta of successful pregnancy decreased in number, and those in miscarried pregnancy increased. In conclusion, the success of the immunization treatment for habitual abortion depends on how to suppress NK cell activity in a linkage with the helper T-cell.

Analysis of ultrastructural immune deposits in passive heymann nephritis by double-staining methods with electron microscopy.

In this study, we located and quantitated the ultrastructural glomerulus-bound immunoglobulins of passive Heymann nephritis from 10 min through 64 days using the double immunogold staining method. At the early stage (10 min to 21 days), rabbit IgG was found predominantly comparing rat IgG. With time (from 21 days to 32 days), rat IgG increased, although rabbit IgG was almost the same as at the early stage. At the late stage (64 days), rat IgG was predominantly found in all of the dense deposits on the glomerular basement membrane, whereas rabbit IgG was decreased. Thus, the double staining method was useful to analyze the composition of the immune complex and also to elucidate the immunopathogenesis of immune complex-mediated glomerulonephritis.

[Influence of pregnancy and steroid hormones on interferon production of lymphocytes].

A study was done to investigate the involvement of interferon (IFN) in immunological acceptance of fetal allograft. IFN was induced by stimulating OK-432 from lymphocyte of pregnant and nonpregnant women as well as men. There was no difference in the IFN titer in these groups. In pregnant women there was no difference between trimesters, either. In addition, we examined the influence of sex steroid hormones such as progesterone and estrogen on IFN production by the lymphocytes of women and men. Generally, each steroid hormone suppressed the production of IFN. However, the extent of suppression varied with the concentration of hormones, resulting in different suppression curves for men and women in a physiological concentration. Differences in response by men and women were also noticed when interleukin 2 was added to examine whether it could reduce the suppression by steroid hormone. This unique sensitivity of female lymphocyte to steroid hormones may play a role in successfully taking a fetal allograft in pregnancy.