Isocyanides Derived from α,α-Disubstituted Amino Acids: Synthesis and Antifouling Activity Assessment.

Herein, we contribute to the development of environmentally friendly antifoulants by synthesizing eighteen isocyanides derived from α,α-disubstituted amino acids and evaluating their antifouling activity/toxicity against the cypris larvae of the Balanus amphitrite barnacle. Almost all isocyanides showed good antifouling activity without significant toxicity and exhibited EC50 values of 0.07 - 7.30 µg/mL after 120-h exposure. The lowest EC50 values were observed for valine-, methionine-, and phenylalanine-derived isocyanides, which achieved > 95% cypris larvae settlement inhibition at concentrations of less than 30 µg/mL without exhibiting significant toxicity. Thus, the prepared isocyanides should be useful for further research focused on the development of environmentally friendly antifouling agents.

Optical assessment of motoneuron function in a "twenty-four-hour" acute spinal cord slice model from fetal rats.

In acute slice preparations of most brain regions, neuronal functions are preserved for only few hours. Since the effects of growth factors or neurotoxic agents are often manifested beyond this time scale, corresponding studies are typically performed on cultured cells. However, cell cultures are generated and maintained under vastly different conditions that can grossly alter neuronal properties. For example, glutamate application to motoneuronal cultures has been reported to modulate neurite formation in some studies while in others it has been reported to kill cells. Here, we have examined whether acute spinal cord slices from rat fetuses can be used within a time window of 24 h for assessment of long-term effects of neuromodulators. In these slices, we have studied the action of glutamate on lumbar motoneurons loaded with fura-2 and rhodamine-123 to monitor intracellular Ca2+ ([Ca2+]i) and mitochondrial potential (Deltapsi), respectively. Further, loading with fura-2 or propidium iodide allowed for morphological assessment of cell viability and death, respectively. Pulses (15 s) or 1 h application of glutamate (300 microM) evoked a moderate (approximately 500 nM) [Ca2+]i rise, but no change of Deltapsi. Even after 24 h, no glutamate-induced cell death was observed and glutamate pulse-evoked [Ca2+]i transients were comparable to controls. The data demonstrate that glutamate does not deregulate [Ca2+]i homeostasis in fetal motoneurons in situ. We propose that acute spinal cord slices from perinatal rodents are a robust model that allows for analysis of neuronal properties and cell viability within a time window of at least 24 h.

Comparison of kinetic and end-point microdensitometry for the direct quantitative histochemical assessment of cytochrome oxidase activity.

Cytochrome oxidase activity has been assessed by a method of kinetic microdensitometry which involves applying tissue sections to gel films containing phenylamine substrates and measuring the rate of azine dye production by continuously recording the rate of change in extinction. Optimum conditions for the technique were defined, and the results compared with those obtained by conventional end-point microdensitometry in which sections are incubated in histochemical substrate solutions and azine dye production estimated by a single measurement of extinction at the end of the incubation period. When compared with biochemically-determined enzyme activity, kinetic microdensitometry gave a better index of the proportionate activity of cytochrome oxidase in various normal tissues than did end-point microdensitometry. In addition, the degree of inhibition of cytochrome oxidase activity in tissues removed from cyanide-poisoned animals was assessed more reliably by kinetic microdensitometry than by end-point measurements. With end-point microdensitometry, the reaction is non-linear over the comparatively long incubation times required and there is also a spontaneous reactivation of cyanide-inhibited cytochrome oxidase during incubation and thus a progressively increased rate of substrate utilization. In contrast, with kinetic microdensitometry the initial linear reaction rate is measured before significant reactivation occurs. Kinetic microdensitometry can be used for direct dynamic quantitation of enzyme activity in tissues or cells; it may be a valuable technique for quantitative histochemical confirmation or extension of biochemical studies; and it appears to be a reliable direct quantitative histochemical method for investigating in vivo inhibition of enzyme activity, where spontaneous reactivation of the enzyme-inhibitor complex may occur.