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pboldSignificance:/boldSide effects of many cancer treatments are associated with the production of reactive oxygen species (ROS) in normal tissues. This explains why patients treated by photodynamic therapy (PDT) often suffer from skin photosensitization, whereas those subject to radiotherapies frequently experience damages in various organs, including the skin./ppboldAim:/boldCatechin, which belongs to the natural flavanols family, is well known for its antioxidant properties. Hence, our main objective was to investigate whether catechin can reduce damages induced by PDT using protoporphyrin IX (PpIX-PDT), an endogenous photosensitizer commonly used in dermatology./ppboldApproach:/boldAnitalicin vivo/italicmodel, the chick embryo chorioallantoic membrane (CAM), was used for this study. An amount of 20 μl of a solution containing 5-aminolevulinic acid, a natural precursor of PpIX, was applied topically on the CAM 4 h before PDTs (405 nm, 2.9 mW / cmsup2/sup, 1.2 J / cmsup2/sup). Catechin was applied at different concentrations (1 to 50 μM) and times (0 to 240 min) before PDT. In addition, we assessed the potency of catechin to reduce the PpIX fluorescence photobleaching induced by PDT./ppboldResults:/boldWe observed that catechin significantly reduces the vascular damages generated by PpIX-PDT. Moreover, we have shown that catechin inhibits PpIX photobleaching./ppboldConclusions:/boldThese observations suggest that catechin significantly reduces the level of ROS produced by PpIX-PDT./p.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture. The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

We describe a simple method of chick whole-embryo culture, which uses a filter paper carrier to hold the early blastoderm and vitelline membranes under tension while the embryo grows on a substratum of agar-albumen. This is a quick and efficient means of setting up cultures of chick embryos beginning at pre-primitive streak stages to stage 10 (stages X--XIV, Eyal-Giladi and Kochav [1976] Dev Biol 49:321-337; stages 1--10, Hamburger and Hamilton [1951] J Morphol 88:49--92). This is an improvement on the original method of New, which used a glass ring and watch glass (New [1955] Exp Morphol 3:320--331). Our modification of New's method, which we call EC (Early Chick, pronounced EASY) culture, facilitates several manipulations in early chick embryos, including microsurgery, grafting, bead implantation, microinjection, and electroporation. Using the EC method, embryos at stage 8 and older can be readily cultured either dorsal-side up (in contrast to New's method) or ventral-side up, as desired; embryos younger than stage 8 can be culture only ventral-side up (as with New's method). We also discuss some alternative methods for setting up these cultures.

During normal development, the neural cell adhesion molecule N-CAM changes at the cell-surface from a sialic acid-rich embryonic, or E form, to several adult, or A forms that have less sialic acid (E-to-A conversion). To investigate the cellular and molecular mechanisms that underlie these changes, we have established conditions under which E-to- A conversion occurs in cultured explants of central nervous system tissues. Mouse cerebellum, chick spinal cord, and chick retina that express the E form of N-CAM were dissected and cultured on collagen gels. After 3-6 d in culture, increased proportions of A forms were synthesized, as revealed by specific immunoprecipitation and immunoblotting. The rate of E-to-A conversion and the proportions of the different A forms synthesized in vitro were similar to those observed for the tissues in vivo at comparable times. In addition, the explants incorporated radioactive precursors of amino sugars into N- CAM, and the electrophoretic mobilities of the E and A forms of N-CAM were altered by treatment with neuraminidase in a way comparable to that found for N-CAM obtained directly from tissue. These results suggest that the post translational processing in vitro was similar to that in vivo. Logistic studies on cell division and death in the explants suggested that E-to-A conversion resulted mainly from a specific increase in synthesis of A forms in individual cells rather than as a consequence of differential birth or death within distinct cell populations. The data were consistent with the possibility that the increase in synthesis of A forms occurred either in cells that had previously synthesized E forms or in a distinct population of cells that already synthesized A forms. Cells dissociated from embryonic central nervous system tissues and cultured in vitro were also found to undergo E-to-A conversion at the same rate as the explant cultures, which suggests that if intercellular signals were responsible for initiation of the change in synthetic pattern, they had already occurred in vivo before the time of culture. In pulse-chase experiments, the E form of N-CAM that was synthesized during the first day after explantation persisted as E form for several days, at times when newly synthesized N-CAM was predominantly in A forms. These results indicate that in cultured neural tissue, the E form of N-CAM is not processed into A forms but is gradually degraded and replaced by newly synthesized A forms.(ABSTRACT TRUNCATED AT 400 WORDS)

Bone and dentine collagens are virtually insoluble in the solvents employed to extract native tropocollagen from soft tissue collagens. The decrease in the ease of extraction of the tropocollagen is generally considered to be due to an increase in the presence of crosslinked components ( see Piez, 1968 ; Bailey, 1968a for reviews ). Bone and dentine collagen must therefore be extensively inter molecularly crosslinked, but the chemistry of the crosslink is unknown. Glimcher and Katz, 1965 , Glimcher et al., 1965 suggested that non-covalent bonds are involved in the stabilization of bone. However a recent report by Miller et al. (1967) clearly demonstrated the presence of stable covalent inter - chain crosslinks in the denatured guanidine-HCl extract from bone. Veis and Schlueter (1964) similarly concluded that dentine was extensively crosslinked by stable covalent bonds, and proposed phosphatemediated ester bonds in addition to a system of periodate sensitive bonds. Our recent studies on the crosslinks of soft tissue collagens have demonstrated the presence of both labile and stable inter molecular crosslinks ( Bailey, 1968b ; Bailey and Peach, 1968 ). The present communication describes the isolation and identification of two covalent inter-chain crosslinks stabilizing bone and dentine.

NG, N G - Dimethylarginine ( unsym - DMA ) has now been found to be present in myosin prepared from developing leg muscle. Neither monomethylarginine nor sym - DMA (NG N ′ G - dimethylarginine ) could be detected in our preparations. Cultured muscle cell myosin contains up to four residues of this amino acid per 5 × 105 grams of protein. Myosins from leg muscle of chick embryos and neonatal rats contained less than two residues of unsym - DMA , while none was detected in adult chicken or rat myosins. Cardiac myosins from developing as well as mature animals lacked or contained very little unsym - DMA . Actin was devoid of this amino acid.

Retinal pigmented epithelial cells of chicken have circumferential microfilament bundles (CMBs) at the zonula adherens region. Isolated CMBs are polygons filled with a meshwork composed primarily of intermediate filaments; they show three major components of 200 000, 55 000, and 42 000 daltons in SDS-gel electrophoresis. Here we have characterized the 55 000-dalton protein immunochemically and ultrastructurally. Immunoblotting and immunofluorescence microscopy have shown that the 55 000-dalton protein is an intermediate filament protein, vimentin. Vimentin filaments changed their distribution during differentiation of pigmented epithelial cells in culture. The protein in the elongated cells showed a fibroblast-type pattern of intermediate filaments. During epithelium formation, the filaments were uniformly distributed and formed a finer meshwork at the apical level. In pigmented epithelial cells that differentiated and matured in culture, vimentin and actin exhibited their characteristic behavior after treatment with colcemid. In the central to basal region of the cell, intermediate filaments formed thick perinuclear bundles. In the apical region, however, intermediate filaments changed in organization from a nonpolarized meshwork to a polarized bundle-like structure. Simultaneously, new actin bundles were formed, running parallel to the intermediate filaments. This suggests that there is some interaction between microfilaments and intermediate filaments in the apical region of these cells.

A monoclonal antibody (C-315) specific for cardiac-type C-protein was prepared and, in combination with other antibodies specific for fast and slow skeletal muscle C-proteins, it was used to investigate the expression of C-protein isoforms in developing striated muscle cells in vivo and in vitro. During embryonic development of skeletal muscles, a C-protein recognized by C-315 appeared first but only transiently, it being replaced subsequently by two other isoforms recognized by the antibodies to slow and fast skeletal muscle C-proteins in a fiber-type specific manner as previously demonstrated (Obinata et al. (1984) Develop. Biol. 101, 116-124). In contrast, only cardiac-type C-protein was detected in cardiac muscle throughout the developmental stages. When myogenesis in vitro was monitored using the same antibodies, C-315 binding appeared first in multinucleated myotubes as in vivo which was followed by the sequential expression of two other C-protein variants. The reactivity of C-315 as well as that of anti-slow and anti-fast skeletal C-protein antibodies persisted during muscle development in culture. Thus, this study demonstrates that the earliest form of C-protein expressed in striated muscles may either be a cardiac-type isoform or a unique embryonic protein containing an epitope in common with the adult cardiac-type protein, and that transitions of C-protein isoform expression characteristic of each fiber-type occur during muscle development in vivo but not in vitro.

The time course of the appearance of myelin-specific markers was studied in the developing chick central nervous system (CNS). Chick CNS tissue was studied for the presence of both proteolipid and myelin basic protein by electroblotting and for 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) by enzyme assay. Four regions of chick spinal cord (cervical, brachial, thoracic and lumbar), brain stem, cerebellum, optic nerve and cortex were studied. In general, myelin basic protein appeared approximately 1 day earlier than proteolipid. In spinal cord and brain stem, myelin basic protein appeared at 13 days incubation. In cerebellum and optic nerve, it appeared at 17 days incubation and in cortex at hatching. CNPase activity increased in most CNS regions between 16 days incubation and hatching. These results suggest that myelination occurs earlier in the chick than in the rat and that it occurs over a shorter time period.