An alpaca single-domain antibody (VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens.

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.

Sign inversion of magnetic circularly polarized luminescence in Iridium(iii) complexes bearing achiral ligands.

Optically inactive, paramagnetic Ir(iii)(ppy)3 and Ir(iii)(ppy)2(acac) (ppy: 2-phenylpyridinate and acac: acetylacetonate) showed nearly mirror-symmetric magnetic circularly polarised luminescence (MCPL) spectra in dilute dichloromethane and dimethyl sulfoxide under N-up and S-up geometries in a 1.6-T magnetic field. However, the MCPL signs of Ir(iii)(ppy)3 and Ir(iii)(ppy)2(acac) under the same N-up (or S-up) Faraday geometry were opposite to each other when one ppy was replaced with an acac. This ligand exchange approach provides facile control of the MCPL sign, irrespective of the Faraday geometry.

Temperature-Dependent Circularly Polarized Luminescence Measurement Using KBr Pellet Method.

Circularly polarized luminescence (CPL) spectroscopy measures the difference in luminescence intensity between left- and right-circularly polarized light, and is often used to analyze the structure of chiral molecules in their excited state. Recently, it has found an increasing range of applications in the analysis of molecules that emit circularly polarized light and can be employed in 3D displays. Thus, the number of articles focusing on CPL spectroscopy has increased dramatically. However, since the luminescence dissymmetry factor (g lum) for organic compounds is generally <|0.01|, CPL spectrometers must offer high sensitivity and produce spectra that are artifact-free for chiral molecules. Until now, the principal targets of CPL measurements have been solution samples. However, for practical device applications, it is also necessary to be able to measure the CPL spectra of solid-state samples. In addition, since electronic devices often operate at high temperatures, it is important to evaluate the thermal dependence of the CPL characteristics. Moreover, in the measurement of solid-state samples, the degree of anisotropy of the samples must be evaluated, because a large degree of anisotropy can cause artifacts. Therefore, we describe methods to evaluate the degree of anisotropy of solid-state samples and their high-temperature applications.

Mirror-image magnetic circularly polarized luminescence (MCPL) from optically inactive EuIII and TbIIItris(ß-diketonate).

Five optically inactive EuIII(hfa)3, TbIII(hfa)3, EuIII(acac)3·Phen, TbIII(acac)3·Phen and EuIII(hfa)3·BDPB (hfa: hexafluoroacetylacetonate, acac: acetylacetonate, BDPB: 2,2'-bis(diphenylphosphino)biphenyl and Phen: phenanthroline) complexes showed clear mirror-image magnetic circularly polarised luminescence (MCPL) spectra in CHCl3, acetone, and DMF solutions, in a poly(methyl methacrylate) film, a KBr pellet and bulk powder at room temperature under 1.6 T as an external magnetic field with Faraday geometry. The signs of the MCPL signals were determined via N-up and S-up geometries. The gMCPL values of EuIII(hfa)3 and TbIII(hfa)3 were ±(0.046-2.0) × 10-2 T-1 at the 5D0 → 5FJ transitions (J = 1-4) in the range of 590 nm and 703 nm and ±(0.0081-1.0) × 10-2 T-1 at the 5D4 → 7FJ transitions (J = 3-6) in the range of 480 nm and 630 nm, respectively. Interestingly, the photoluminescence spectra of EuIII(hfa)3 and TbIII(hfa)3 in CHCl3 and acetone under 1.6 T considerably redshifted by 10-114 cm-1, while those of EuIII(acac)3·Phen and TbIII(acac)3·Phen did not reveal detectable redshifts.

Equation for calculating the concentration of solvent in air that discriminates between exposure and non-exposure based on biomarker concentrations in the urine of workers.

To develop a new method for evaluating the intensity of workers. exposures to toluene alone or toluene in mixed solvents, regression equations were calculated between the concentrations of toluene to which workers were exposed and the concentrations of hippuric acid or toluene in workers. urine samples taken at the end of their shifts. Thereafter, the discriminant exposure concentration of the solvents in air, which was the concentration considered to discriminate exposure from non-exposure within a fixed level of error using fiducial ranges of individual specimens (DEC-I) or using confidence ranges of regression equation (DEC-R), was measured by a scale. The devised equations were applied to calculate DEC-I or DEC-R accurately using the formulas expressing a regression line and its fiducial ranges or confidence ranges. The equations can calculate not only more precise values of DEC-I or DEC-R than can be measured by a scale, but can also calculate values corresponding to any level of error. Moreover, DEC-I and DEC-R can be defined by the equations. The concentration capable of discriminating TLV (threshold limit value) exposure from non-TLV exposure was estimated using fiducial ranges (DTL-I) and then using confidence ranges of the regression equation (DTL-R).

Effect of antifungal azoles on the heme detoxification system of malarial parasite.

The antimalarial activities of some antifungal azole agents (ketoconazole, miconazole, and clotrimazole) have been known for several years, however, their antimalarial mechanism remains equivocal. Our recent study showed that clotrimazole has a relative high affinity for heme, inhibits reduced glutathione-dependent heme catabolism, and enhances heme-induced hemolysis. In the present study, we have found that clotrimazole can remove heme from histidine rich peptide-heme complex, which initiates heme-polymerization in malaria. In addition, we show that two other azoles (ketoconazole and miconazole) behave similarly to clotrimazole in binding to heme: they bind to heme with similar affinities, remove heme from the histidine rich peptide-heme complex and from the reduced glutathione-heme complex to form stable heme-azole complexes with two nitrogenous ligands derived from the imidazole moieties of two azole molecules. We have also revealed that clotrimazole and miconazole have stronger promoting activities for heme-induced hemolysis than ketoconazole, implying that the stronger antimalarial activities of clotrimazole and miconazole might arise from their stronger ability to promote heme-induced hemolysis of clotrimazole and clotrimazole than that of ketoconazole. These results also suggest that ketoconazole and miconazole, like clotrimazole, might possess an antimalarial mechanism relating to their inhibition of heme polymerization and the degradation of reduced glutathione-dependent heme.

Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2.

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H(2)O(2)-induced degradation of heme in food vacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (K(d) = 2.21 x 10(-6) M and 0.71 x 10(-6) M, respectively) might be as high as that of PfHRP2 (K(d) = 0.94 x 10(-6) M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. Therefore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.

Electrochromic Type E-Paper Using Poly(1H-Thieno[3,4-d]Imidazol-2(3H)-One) Derivatives by a Novel Printing Fabrication Process.

In this study, we report poly(1H-thieno[3,4-d]imidazol-2(3H)-one) (pTIO) derivatives for an electrochromic (EC) type e-paper and its novel printing fabrication process. pTIO is a kind of conductive polymer (CP) s which are known as one of the EC materials. The electrochromism of pTIO is unique, because its color in doped state is almost transparent (pale gray). A transparent state is required to show a white color in a see-through view of an EC type e-paper. An electrochromism of CP has a good memory effect which is applicable for e-paper. The corresponding monomers of CP are able to be polymerized with an electrochemical method, which be made good use of for the fabrication process of e-paper. pTIO derivatives are copolymerized with other pi-conjugated X unit, which adjusts the color of electrochromism. Finally, we fabricated a segment matrix EC display using pTIO derivatives by ink-jet printing.

Clotrimazole binds to heme and enhances heme-dependent hemolysis: proposed antimalarial mechanism of clotrimazole.

Two recent studies have demonstrated that clotrimazole, a potent antifungal agent, inhibits the growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum, in vitro. We explored the mechanism of antimalarial activity of clotrimazole in relation to hemoglobin catabolism in the malaria parasite. Because free heme produced from hemoglobin catabolism is highly toxic to the malaria parasite, the parasite protects itself by polymerizing heme into insoluble nontoxic hemozoin or by decomposing heme coupled to reduced glutathione. We have shown that clotrimazole has a high binding affinity for heme in aqueous 40% dimethyl sulfoxide solution (association equilibrium constant: K(a) = 6.54 x 10(8) m(-2)). Even in water, clotrimazole formed a stable and soluble complex with heme and suppressed its aggregation. The results of optical absorption spectroscopy and electron spin resonance spectroscopy revealed that the heme-clotrimazole complex assumes a ferric low spin state (S = 1/2), having two nitrogenous ligands derived from the imidazole moieties of two clotrimazole molecules. Furthermore, we found that the formation of heme-clotrimazole complexes protects heme from degradation by reduced glutathione, and the complex damages the cell membrane more than free heme. The results described herein indicate that the antimalarial activity of clotrimazole might be due to a disturbance of hemoglobin catabolism in the malaria parasite.