Time and space-resolved quantification of plasma membrane sialylation for measurements of cell function and neurotoxicity

While there are many methods to quantify the synthesis, localization, and pool sizes of proteins and DNA during physiological responses and toxicological stress, only few approaches allow following the fate of carbohydrates. One of them is metabolic glycoengineering (MGE), which makes use of chemically modified sugars (CMS) that enter the cellular biosynthesis pathways leading to glycoproteins and glycolipids. The CMS can subsequently be coupled (via bio-orthogonal chemical reactions) to tags that are quantifiable by microscopic imaging. We asked here, whether MGE can be used in a quantitative and time-resolved way to study neuronal glycoprotein synthesis and its impairment. We focused on the detection of sialic acid (Sia), by feeding human neurons the biosynthetic precursor N-acetyl-mannosamine, modified by an azide tag. Using this system, we identified non-toxic conditions that allowed live cell labeling with high spatial and temporal resolution, as well as the quantification of cell surface Sia. Using combinations of immunostaining, chromatography, and western blotting, we quantified the percentage of cellular label incorporation and effects on glycoproteins such as polysialylated neural cell adhesion molecule. A specific imaging algorithm was used to quantify Sia incorporation into neuronal projections, as potential measure of complex cell function in toxicological studies. When various toxicants were studied, we identified a subgroup (mitochondrial respiration inhibitors) that affected neurite glycan levels several hours before any other viability parameter was affected. The MGE-based neurotoxicity assay, thus allowed the identification of subtle impairments of neurochemical function with very high sensitivity.

Influence of growth regulators and respiration inhibitors on dark transformation of phytochrome in coleoptiles of oat seedlings

Irradiation with red light leads to the formation of an unstable, undergoing gradual destruction, physiologically active P_FR form of phytochrome in the coleoptiles of oat seedlings. Growth substances: IAA, GA₃, kinetin, ABA, ethrel as well acetylcholine do not influence the nature and rate of phytochrome dark transformation. Inhibitors of energy-producing processes such as KCN, 2,4-DNP, DCCD and antimycin A inhibit the process of dark destruction of the P_FR form of phytochrome.

Endogenous oscillations of electric potential difference in the cambial region of the pine stem. I. General characteristics of the oscillations

Extracellular measurements of electric potential difference (PD) in isolated tissue blocks from Scots pine were performed. Endogenous transient PD changes (oscillations) generated exclusively in the cambial region were found. They were not recorded in tissue blocks exposed to drastic thermal treatment. The oscillations were significantly damped by respiration inhibitors and stimulated by fusicoccin. The results point to the cambial region as the main source of electric activity in a woody stem.