PHOTOREACTIVATION ASSOCIATED WITH CHROMOSOMAL ABNORMALITY IN SEA URCHIN EGGS FERTILIZED WITH ULTRAVIOLET-IRRADIATED SPERM

Abstract— As a model for studies involving biologic systems, rates of hematoporphyrin and protopor-phyrin-sensitized photooxidation of tryptophan were measured in water-methanol mixtures. Absorption and fluorescence-emission spectra associated with solutions of porphyrins in different water-methano! mixtures were also examined. The optimal rate of photooxidation by hematoporphyrin occurred in 30% methanol; by protoporphyrin, in 60% methanol. Absorption spectra were examined to characterize dispersion of hematoporphyrin into monomeric form in 90% methanol, and the dimer formation which predominates in 30% methanol. With the more hydrophobic protoporphyrin, an absorption spectrum characteristic of drug monomer was seen in 100% methanol, a dimer spectrum was observed in 60% methanol. At methanol concentrations < 60%, we observed impaired photooxidation and another alteration in the absorption pattern apparently reflecting drug aggregation beyond the dimer stage. Fluorescence emission maxima of both porphyrins were blue-shifted with increasing solvent dielectric constant, but an additional blue shift was detected in an aqueous environment which was apparently related to porphyrin aggregation. Absorption and fluorescence-emission spectra appear helpful for characterizing environmental determinants of porphyrin-sensitized photooxidation.     

ACTION SPECTRUM FOR PHOTOREACTIVATION OF ULTRAVIOLET-INDUCED MORPHOLOGICAL ABNORMALITY IN SEA URCHIN EGGS

Abstract An action spectrum was obtained for photoreactivation (PR) of morphological abnormality arising from ultraviolet (UV)-irradiation of sea urchin sperm. The wavelength dependence of PR was measured by the restoration of the formation of normal pluteus larvae after the exposure of fertilized eggs to various fluences of monochromatic PR light (313 to 500 nm). The PR action spectrum showed a maximum around 365 nm and a secondary peak somewhere above 400 nm. High PR activity beyond 400 nm wavelengths may reflect an advantageous or adaptational ability to cope with harmful effects of solar UV radiation.     

Preparation of Porous Protein-Based Hydogel for Highly Sensitive Protein Chips

Summary: Protein chips are important tools for high-throughput analysis of biological events. We have developed a novel method to prepare a protein-based hydrogel, that is, a “Three-Dimensional Nano-structured Protein Hydrogel” (3-D NPH), which is composed of protein and polymer nano-particles. The 3-D NPH could be easily prepared by dispensing a protein and polymer mixture on a substrate. Surprisingly, gold particles conjugated with protein A diffused into the 3-D NPH which was made of mouse IgG through the pores. We have shown that the protein chips made with our 3-D NPH method has tremendously improved sensitivity in detecting protein-protein interactions compared with that of direct protein immobilization methods.     

PHOTOREVERSAL OF ABNORMAL MORPHOGENESIS IN SEA-URCHIN EMBRYOS CAUSED BY UV-IRRADIATION

Abstract— Morphological abnormalities induced by UV-irradiation of 8- or 16-cell-stage embryos of the sea-urchin, Hemicentrotus pulchenimus , and their photoreversal were studied. UV-irradiation of the animal hemisphere of embryos caused the formation of exogastrulae, while that of the vegetal hemisphere caused the formation of permanent blastulae. These UV-induced morphological abnormalities were photoreversed when the UV-irradiated embryos were subsequently illuminated with visible light, so that the UV-irradiated embryos developed into normal pluteus-larvae. When UV-irradiated embryos were illuminated with visible light up to the onset of the DNA-synthesis phase of the following cell cycle, the UV-induced morphological abnormalities were photoreversed almost completely. The effectiveness of an exposure to visible light declined thereafter and was subsequently completely lost.     

Activity of enzymes immobilized on microspheres with thermosensitive hairs

Poly(N-isopropylacrylamide)s (PNIPAMs) carboxylated at one chain end or both ends were prepared by polymerization using 4,4-azobis(N,N,-cyanopentanoic acid) (V-501) as an initiator and β-mercaptopropionic acid (MPA) as a chain transfer reagent. One end group of PNIPAM carboxylated at both ends was conjugated with latex particles, and another with trypsin using carbodiimide. Differential scanning calorimetry (DSC) revealed that PNIPAM on the particles exhibited a drastic phase transition, and that the transition temperature was largely elevated when the enzyme was immobilized at the chain end. Therefore, PNIPAM on the particles showed two phase transitions because of the coexistence of the enzyme-conjugated and non-conjugated PNIPAMs. The activity of trypsin immobilized on the particles with the PNIPAM spacer showed significant temperature dependence. The apparent relative activity increased above the transition temperature of non enzyme-conjugated PNIPAM on the particles. One of the reasons for this is that the diffusion of the substrate changed discontinuously around the transition temperature. Therefore, the temperature dependence of the enzymatic activity was significantly affected by the molecular size of the substrates. The enzymatic activity was also influenced by the surface density of trypsin and PNIPAM on the particle, and the molecular weight of the PNIPAM spacer.     

Preparation of colored latex containing oil-soluble dyes with high dye content by mini-emulsion polymerization

Polymer particles containing oil-soluble dyes (colored latex particles) were prepared by mini-emulsion polymerization. Copper phthalocyanine dyes and styryl dyes were used as oil-soluble dyes. Highly hydrophobic dyes played the role of hydrophobe by themselves and enabled the full incorporation of dyes in the latex without additional hydrophobes. Two phthalocyanine dyes having similar color were blended in a mini-emulsion polymerization so that the resulting colored latex showed enough strong color depth as a colorant. Colored latexes with high dye content (more than 30 wt% for phthalocyanine dye system and 40 wt% for styryl one) and with particle size less than 100 nm were obtained.     

Porous Protein‐Based Nanoparticle Hydrogel for Protein Chips with Improved Sensitivity

We have discovered a novel method to prepare a protein‐based hydrogel, that is, a ‘three‐dimensional nanostructured protein hydrogel’ (3D NPH), which is composed of loosely inter‐connected protein–polymer hybrid nanoparticles. The 3D NPH can be easily prepared by spotting a protein/polymer mixture on a substrate. Surprisingly, gold nanoparticles carrying protein molecules easily diffuse into the 3D NPH through pores and spaces. We have shown that the protein chip made by our 3D NPH method has tremendously improved sensitivity in detecting protein–protein interactions compared with that by direct protein immobilization methods.

     

Improvement of the storage stability and photostability of colored latex prepared by miniemulsion polymerization

Polymer particles containing oil-soluble dyes (colored latex) were prepared by miniemulsion polymerization. The dye preservation properties of colored latex loaded with different types of dye were studied; preservation depended on the rate of dye diffusion and thus on the size of the dye molecules and the hardness of the polymer matrix in the latex particles. Hindered amine stabilizers (HAS) were used to improve the photostability of colored latex. The effect of HAS in colored latex was investigated both as an additive and as a comonomer. The photostability of latex particles containing a simple mixture of dye and HAS was improved with increasing HAS content up to 20% in particles. In the case of colored latex particles combined with HAS by a covalent bond, the optimal amount of HAS for photostability improvement was between 8 and 20%.