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Results are reported on the sensitivity of various gynaecological tumour cell lines to 5-aminolaevulinic acid-induced protoporphyrin IX-sensitised photodynamic therapy (ALA-PDT) in vitro. All cell lines tested accumulated ALA-induced protoporphyrin IX (PpIX) and demonstrated good sensitivity to ALA-PDT. Localisation of PpIX in the mitochondria was demonstrated by fluorescence microscopy. Subcellular damage following ALA-PDT was observed using transmission electron microscopy. This damage was localised initially to the mitochondria, with damage to membranes and the nucleus and complete loss of intracytoplasmic organisation being observed subsequently. There was no apparent difference in ALA-PDT response between a multidrug-resistant ovarian carcinoma cell line and its parent line. These results indicate that ALA-PDT has potential for application to therapy of gynaecological malignancies. Images Figure 5 Figure 6 Figure 7

Specific, straightforward, and rapid procedures are required for the detection, identification, and quantification of the potent low molecular weight toxins that are produced by blooms and scums of cyanobacteria (blue-green algae) in waterbodies. Use of the Microtox bioluminescence assay and the brine shrimp (Artemia salina) has been advocated for the initial screening of cyanobacterial blooms for microcystin hepatotoxins. Inhibition of bacterial luminescence in the Microtox assay and brine shrimp mortality were determined with microcystin-containing and nonmicrocystin-containing cyanobacteria. Extraction and fractionation of test samples was undertaken to select and isolate microcystincontaining fractions and reduce interference from other fractions. Maximal inhibition of bacterial luminescence in the Microtox assay occurred with fractions from Microcystis strains and an Anabaena bloom that did not contain microcystins. By contrast, the bioassay of fractions using brine shrimps correlated with the distribution of microcystin-LR in the fractionated Microcystis extracts. © 1994 by John Wiley & Sons, Inc..

Smooth muscle caldesmon was phosphorylated in vitro by sea star p44mpk up to 2.0 mol of phosphate/mol of protein at both Ser and Thr residues. The phosphorylation sites were contained mainly in the COOH-terminal 10-kDa cyanogen bromide fragment which houses the binding sites for calmodulin, tropomyosin, and F-actin. Tryptic peptide maps of 32P-labeled caldesmon by p44mpk and p34cdc2 showed that while both enzymes recognized similar sites of phosphorylation, they have different preferred sites. Phosphorylation of caldesmon attenuated slightly its interaction with actin and had no effect on its binding to calmodulin and tropomyosin. Smooth muscle cell extracts from chicken gizzard and rat aorta contained 42- and 44-kDa proteins, respectively, which were cross-reactive with an antibody to sea star p44mpk. Immunoprecipitates from gizzard and aorta cell extracts, generated with the p44mpk antibody, possessed kinase activities toward myelin basic protein as well as caldesmon. These results suggest that MAP kinase may have functions in the differentiated smooth muscle cells distinct from those involved in the cell cycle.

Vascular mediator synthesis in endothelial cells is Ca 2+ sensitive. Bradykinin increases [Ca 2+ ] i by releasing it from intracellular stores and by increasing influx across the plasmalemma. The latter is believed to occur through receptor-operated channels. Although gating of these plasmalemmal channels is voltage-insensitive, we hypothesized that Ca 2+ influx would still be dependent on the Ca 2+ electrochemical gradient and relative cation permeability. Using cultured bovine pulmonary endothelial cells we therefore measured: membrane voltage ( E m ) in single cells using the “tight seal” whole cell recording technique, Ca 2+ i in endothelial cell monolayers using fura-2, and arachidonic acid (AA) release using 3 H-AA prior to and following exposure to bradykinin at different [K + ] 0 . Our data indicate that the resting membrane potential of these cells is at least −67 mV in physiological saline and that the background resting membrane properties can be described with a ( P Na P K ) ratio of ∼0.027–0.040. Varying [K + ] 0 is shown to be an effective means for altering and controlling membrane potential and thus the calcium electrochemical gradient. Increases in [K + ] 0 lead to a concentration-dependent decrease in the magnitude of the Ca 2+ transient and in the relative amount of arachidonic acid released following exposure to bradykinin suggesting that Ca 2+ influx through the plasmalemma and AA release are regulated by the Ca 2+ electrochemical gradient. In addition, a simple theoretical membrane conductance model is presented which is able to reconcile the wide range in apparent resting membrane potentials which have been reported for endothelial cells.

A simple flow cytometric technique for rapid measurement of multilog cytotoxic responses to photosensitization of cellular systems is described. This technique is particularly useful for cell lines with a low colony-forming efficiency, for which a nonclonogenic assay is required. The assay separates cell-sized objects from cellular debris by gating on forward scatter versus side scatter, identifies viable cells by positive calcein AM and negative ethidium homodimer-1 staining and measures cell concentration relative to an internal standard of polystyrene beads. Large numbers of cells can be analyzed rapidly. Two patient-derived small cell lung cancer cell lines, NCI-H209 and SV-E, were used to test the technique. Photordiation survival curves of the response of these cell lines to 5-aminolevulinic acid-induced protoprophyrin IX photosensitization correlated with the extent of photosensitizer accumulation. There was good agreement between the results obtained using the tritiated thymidine incorporation assay and the flow cytometric cytotoxicity assay. The technique can be used to measure cytotoxic responses to photosensitization of cell lines regardless of their plating efficiencies.

The recent rise in the awareness of the occurrence of toxic cyanobacterial blooms in aquatic environments, with associated human health problems and animal deaths, has increased the need for rapid, reliable and sensitive methods of determining cyanobacterial toxicity. A luminescent bacterial toxicity test was assessed as a complement to the established mouse bioassay. Seventeen samples including pure cyanobacterial microcystin-LR hepatotoxin, laboratory isolates and natural blooms of cyanobacteria were tested and toxicity data compared with mouse LD50 values. Microcystin-LR and all five microcystin-containing cyanobacterial samples, hepatotoxic by mouse test gave EC50 values of less than 0.46 mg/ml in bioluminescence-based Microtox assays. Of 11 samples non-toxic by mouse bioassay, only two gave an EC50 of less than 0.98 mg/ml by bioluminescence assay. It is suggested that the Microtox bioluminescence assay may prove useful in the preliminary screening of cyanobacterial blooms for microcystin-based toxicity.

Previous models of cardiac cellular electrophysiology have been based largely on voltage-clamp measurements obtained from multicellular preparations and often combined data from different regions of the heart and a variety of species. We have developed a model of cardiac pacemaking based on a comprehensive set of voltage-clamp measurements obtained from single cells isolated from one specific tissue type, the bullfrog sinus venosus (SV). Consequently, sarcolemmal current densities and kinetics are not influenced by secondary phenomena associated with multicellular preparations, allowing us to realistically simulate processes thought to be important in pacemaking, including the Na(+)-K+ pump and Na(+)-Ca2+ exchanger. The membrane is surrounded extracellularly by a diffusion-limited space and intracellularly by a limited myoplasmic volume containing Ca2(+)-binding proteins (calmodulin, troponin). The model makes several predictions regarding mechanisms involved in pacing. 1) Primary pacemaking cannot be attributed to any single current but arises from both the lack of a background K+ current and a complex interaction between Ca2+, delayed-rectifier K+, and background leak currents. 2) Ca2+ current displays complex behavior and is important during repolarization. 3) Because of Ca2+ buffering by myoplasmic proteins, the Na(+)-Ca2+ exchanger current is small and has little influence on action potential repolarization but may modulate the maximum diastolic potential. 4) The Na(+)-K+ pump current does not play an active role in repolarization but is of sufficient size to modulate the rate of diastolic depolarization. 5) K+ accumulation and Ca2+ depletion may occur in the extracellular spaces but play no role in either the diastolic depolarization or repolarization of a single action potential. This model illustrates the importance of basing simulations on quantitative measurements of ionic currents in myocytes and of including both electrogenic transporter mechanisms and Ca2+ buffering by myoplasmic Ca2(+)-binding proteins.

Passive integrated transponder (PIT) tags were injected into 22 largemouth bass (Micropterus salmoides) brood fish to determine the retention rate of the tags, the effect on spawning success, and the utility of the tags as a means of individual fish identification. Fish were evaluated 12, 17, and 24 months after implantation. All tags were retained and all tagged fish were recognized. Tag injection and retention had no discernible effect on spawning success.

1. Experimental measurements and computer simulations have been used in attempts to identify an exchanger current (Iex) generated by an electrogenic Na+-Ca2+ exchanger in single cells from bull-frog atrium. 2. Voltage clamp measurements of an inward 'slow tail' current observed upon repolarization after depolarizing clamp pulses that elicit net inward Ca2+ currents (ICa) (see Campbell, Giles & Shibata, 1988c), show that these slow tails have a cationic dependence different from ICa. Slow tails are large and prominent in normal [Na+]o solutions containing either Ca2+ or Sr2+, but they are markedly reduced or absent in Ba2+, Ca2+-free, and Na+-free solutions. 3. Kinetic measurements on the slow tails show that they are not generated by deactivation of ICa, and suggest that they may be due to activation of Iex at negative potentials (-70 to -100 mV). 4. Computer simulations of the influx, buffering, and extrusion of Ca2+ provide further indirect evidence that the slow tails correspond to Iex. In addition, these calculations give insights into one plausible mechanism of Ca2+ homeostasis in frog atrium. When the Na+-Ca2+ exchanger formalism of Mullins (1979, 1981), as modified by DiFrancesco & Nobel (1985), is combined with equations for intracellular Ca2+ buffering by myoplasmic proteins (cf. Robertson, Johnson & Potter, 1981), slow inward tails are produced which are qualitatively similar to those recorded experimentally. 5. Comparisons of the size and time course of ICa with those of Iex suggest that Iex does not generate a physiologically significant current (or membrane potential change) during the plateau of the action potential. However, at potentials near the resting potential the inward current due to Iex may be significant. 6. Our theoretical results suggest that in the intact single atrial cell myoplasmic Ca2+-binding proteins (e.g. calmodulin and troponin) could be physiologically important modulators of the amplitude, polarity and kinetics of Iex. Hence, the specificity, capacity and kinetics of intracellular Ca2+ binding are essential components of any quantitative treatment of Iex in excitable tissue.