Caged probes: a novel tool in studying symplasmic transport in plant tissues

Summary. Caged probes offer a novel approach to study plant cell-to-cell communication. Instead of introducing fluorescent molecules into cells by microinjection, their caged counterparts can be preloaded into the tissue by diffusion. Following spatially controlled photoactivation, movement of the uncaged fluorochrome can be followed in time and direction by confocal laser scanning microscopy. In the onion bulb scale epidermis used as a model system, symplasmic transport of the tracer out of a target cell was followed. Transport via the symplasmic pathway was challenged by plasmolysing the tissue. The experiments confirmed the symplasmic nature of tracer transport.Correspondence and reprints: Department of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C. Denmark. E-mail: hjm@kvl.dk     

Synthesis and enzymatic incorporation of photolabile dUTP analogues into DNA and their applications for DNA labeling

Two novel photolabile nucleotide triphosphate (NTP) analogues were synthesized through Sonogashira coupling and their enzymatic incorporation into DNA was evaluated with three different DNA polymerases (Taq, Vent exo- and T4) by polymerase chain reaction. Both nucleotide triphosphate analogues were recognized by these DNA polymerases as substrates for primer extension. Light irradiation of PCR products removed the photolabile group and released the amino and carboxyl moieties. Further site-specific dual-labeling for oligodeoxynucleotides (ODNs) and random labeling for a long DNA construct with fluorophores were successfully achieved with incorporation of the photolabile amine modified deoxyuridine triphosphate (dUnTP).     

PhotoCORMs: Light-triggered release of carbon monoxide from the coordination sphere of transition metal complexes for biological applications

Carbon monoxide is now well-established as a small-molecule biological effector in the human body. Metal-carbonyl complexes are a promising way to achieve safe and controlled delivery of CO for therapeutic applications and thus, such CO releasing molecules (CORMs) have achieved significant attention in the last 10 years. In most CORMs, the liberation of carbon monoxide is triggered by hydrolytic processes in aqueous medium and thus their half-life under physiological conditions determines their potential therapeutic utility. To overcome such limitations, photo-induced CO release from dark-stable metal-carbonyl complex prodrugs is an interesting alternative. Thus, in this review, the current knowledge on PhotoCORMs is summarized and their properties critically evaluated. The main challenge for the future will be to achieve photolytic liberation of carbon monoxide by near-IR excitation in the phototherapeutic window of the cell. Different ways how this goal might be achieved are discussed.     

Requirement of divalent cations for photoactivation of the laten water-oxidation system in intact chloroplasts from flashed leaves

The effects of divalent cations on photoactivation of the latent water-oxidation system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination were investigated by using A23187, an ionophore for divalent cations, and the following results were obtained. (a) Photoactivation in the intact chloroplasts was inhibited by A23187, but was restored on addition of a low concentration of Mn²⁺ (10 μM). (b) A high concentration of Mn²⁺ (70 μM) was inhibitory, in contrast, for photoactivation, but the inhibition was restored by the coexistence of a suitable concentration of Ca²⁺ (5 mM). (c) The Ca²⁺-dependent restoration was inhibited by a high concentration of Mg²⁺ or Sr²⁺, but the inhibition was restored by the coexistence of Ca²⁺. (d) Kinetic analyses of these competitive effects between divalent cations revealed that: (i) High concentration of Ca²⁺ inhibits photoactivation in competition with Mn²⁺. (ii) High concentration of Mn²⁺ inhibits photoactivation in competition with Ca²⁺. (iii) High concentration of Mg²⁺ affects photoactivation by inhibiting Ca²⁺-dependent restoration in competition with Ca²⁺. Based on these results, we propose that the latent water-oxidation center has two binding sites, each specific for Mn²⁺ and Ca²⁺, and that photoactivation takes place in the center having both Mn²⁺ and Ca²⁺ on their respective binding sites.