Retinal vasculogenesis and ischemic retinopathies provide good model systems for study of vascular development and neovascularization (NV), respectively. Vascular endothelial cell growth factor (VEGF) has been implicated in the pathogenesis of retinal vasculogenesis and in the development of retinal NV in ischemic retinopathies. However, insulin-like growth factor-I and possibly other growth factors also participate in the development of retinal NV and intraocular injections of VEGF antagonists only partially inhibit retinal NV. One possible conclusion from these studies is that it is necessary to block other growth factors in addition to VEGF to achieve complete inhibition of retinal NV. We recently demonstrated that a partially selective kinase inhibitor, PKC412, that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors and several isoforms of protein kinase C (PKC), completely inhibits retinal NV. In this study, we have used three additional selective kinase inhibitors with different selectivity profiles to explore the signaling pathways involved in retinal NV. PTK787, a drug that blocks phosphorylation by VEGF and PDGF receptors, but not PKC, completely inhibited retinal NV in murine oxygen-induced ischemic retinopathy and partially inhibited retinal vascularization during development. CGP 57148 and CGP 53716, two drugs that block phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on retinal NV. These data and our previously published study suggest Neovascularization (NV) occurs in wound repair and several pathological processes including tumor growth, arthritis, atherosclerosis, and proliferative retinopathies. Although there are likely to be tissue-specific differences, there are also likely to be shared features, so that new knowledge regarding one of these pathologies may provide insights for the others. Proliferative retinopathies provide good model systems for study of NV, because the new blood vessels can be visualized in vivo and the ocular circulation is well-studied, providing important background information.The retina is a tissue with very high metabolic activity that is oxygenated from retinal and choroidal circulations, which each originate from branches of the ophthalmic artery. The choroidal circulation is derived from the long and short posterior ciliary arteries, which pierce the sclera and form successively smaller branches that supply the choriocapillaris, fenestrated microvessels separated from the retina by the retinal pigmented epithelium (RPE). The photoreceptor layer of the retina has no blood vessels and receives oxygen by diffusion from the cho-

Basic fibroblast growth factor (FGF2) is constitutively expressed in the retina and its expression is increased by a number of insults , but its role in the retina is still uncertain. This study was designed to test the hypothesis that altered expression of FGF2 in the retina affects the development of retinal neovascularization. Mice with targeted disruption of the Fgf2 gene had no detectable expression of FGF2 in the retina by Western blot , but retinal vessels were not different in appearance or total area from wild-type mice. When FGF2-deficient mice were compared with wild-type mice in a murine model of oxygen-induced ischemic retinopathy , they developed the same amount of retinal neovascularization. Transgenic mice with a rhodopsin promoter/Fgf2 gene fusion expressed high levels of FGF2 in retinal photoreceptors but developed no retinal neovascularization or other abnormalities of retinal vessels; in the ischemic retinopathy model , they showed no significant difference in the amount of retinal neovascularization compared with wild-type mice. These data indicate that FGF2 expression is not necessary nor sufficient for the development of retinal neovascularization. This suggests that agents that specifically antagonize FGF2 are not likely to be useful adjuncts in the treatment of retinal neovascularization and therapies designed to increase FGF2 expression are not likely to be complicated by retinal neovascularization. (Am J Pathol 1998, 153:757-765)

Betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) is an important enzyme that catalyzes the last step in the synthesis of glycine betaine, a compatible solute accumulated by many plants under various abiotic stresses. In barley (Hordeum vulgare L.), we reported previously the existence of two BADH genes (BBD1 and BBD2) and their corresponding proteins, peroxisomal BADH (BBD1) and cytosolic BADH (BBD2). To investigate their enzymatic properties, we expressed them in Escherichia coli and purified both proteins. Enzymatic analysis indicated that the affinity of BBD2 for betaine aldehyde was reasonable as other plant BADHs, but BBD1 showed extremely low affinity for betaine aldehyde with apparent K(m) of 18.9 microM and 19.9 mM, respectively. In addition, V(max)/K(m) with betaine aldehyde of BBD2 was about 2000-fold higher than that of BBD1, suggesting that BBD2 plays a main role in glycine betaine synthesis in barley plants. However, BBD1 catalyzed the oxidation of omega-aminoaldehydes such as 4-aminobutyraldehyde and 3-aminopropionaldehyde as efficiently as BBD2. We also found that both BBDs oxidized 4-N-trimethylaminobutyraldehyde and 3-N-trimethylaminopropionaldehyde.

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Volatile compounds, such as beta-cyclocitral, geosmin, and 2-methylisoborneol, from cyanobacteria showed a lytic activity against cyanobacteria. Particularly, beta-cyclocitral caused an interesting color change in the culture broth from green to blue during the lysis process. In the present study, the lytic behavior of various cyanobacteria with beta-cyclocitral was investigated, and a mechanism for the blue color formation was developed. beta-Cyclocitral lysed both the laboratory strains of any genera and bloom samples including many species of cyanobacteria, and caused the characteristic color change from green to blue. beta-Cyclocitral provided a characteristic behavior, such that the absorption maxima of chlorophyll-a and beta-carotene disappeared, but that of phycocyanin still remained after 12 h, which indicated that beta-cyclocitral decomposed chlorophyll-a and beta-carotene rapidly, so that the inherent colors from the tolerant water-soluble pigments became observable in the cultured broth. This phenomenon was confirmed by another experiment using Phormidium (NIES-611), which showed a pink color derived from phycoerythrin. beta-Cyclocitral was more easily oxidized when compared with similar aldehyde compounds, so that the pH of the solution quickly decreased to 4.5. An oxidation product of beta-cyclocitral in water solution was isolated and identified as 2,6,6-trimethylcyclohexene-1-carboxylic acid. This study provides support that beta-cyclocitral derived from cyanobacteria plays an important role in the lysis of cyanobacteria and participates in the blue color formation under natural conditions.

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline → betaine aldehyde → GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [(14)C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [(14)C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.

In a previous study, it was suggested that β-cyclocitral plays an important role in understanding the lysis of cyanobacteria under natural conditions. The present study was conducted in order to understand how the β-cyclocitral lyses cyanobacterial cells, and other anticyanobacterial agents were also investigated. The preliminary study using a scanning electron microscope (SEM) demonstrated that there were three types of morphological changes in the cyanobacterial cells when they were incubated with anticyanobacterial agents: volatile compounds from cyanobacteria cause shrinking and then wrinkling; terpenoids contact directly and cause stripping; basic amino acids cause swelling and then collapsing. In order to clarify the extreme difference in the damage during the morphological changes between the β-cyclocitral and L-lysine (Lys), the transmission electron microscope (TEM) technique was applied. Although a definite difference in the morphological damage was observed, a plausible mechanism for β-cyclocitral could be not deduced. Throughout the experiments using antibiotics, it was found that the apparent morphological changes after lysis did not always correspond to the mode of action.