Cationic liposomes with a cationic cholesterol derivative are known to be useful tools for gene transfection. In the present paper we used atomic force microscopy (AFM) for studying the structure of cationic liposomes complexed with plasmid DNA. Cationic liposomes were made by a mixture of cholesteryl-3β-carboxyamidoethylenedimethylamine (I) and 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE). AFM images of free plasmid DNA (pSV2CAT) clearly showed a circular structure of plasmid DNA with a length of 1.4 μm, and those of free cationic liposomes showed small unilamellar vesicles with diameters of 200 nm. Complex formation of the cationic liposomes with plasmid DNA showed drastic morphological changes of the liposomes. The cationic liposome-DNA complex formed a necklace-like structure at a charge ratio of 0.6 (cholesterol/phosphate) and the complex formed a capsulated structure with a diameter of 1—1.5 μm at a charge ratio of 1.2, which ratio was the most appropriate for gene transfection. Thus, it seemed that this kind of capsulation was important for gene transfection mediated by the cationic liposomes.

抄録全体を表示

The uptake of 3'-cholesterol conjugated phosphorothioate oligonucleotides with 5'-fluorescein (ChPSf-ODN) into rat immortalized hepatocytes was visualized with a real-time confocal laser microscope. The images showed that the uptake of ChPSf-ODN in the cytosol had started within the first 5 minutes, and it was not until a 40 minute-incubation that the endosomal and/or lysosomal accumulation appeared. Histogram analysis of fluorescence intensity indicated that cytosolic uptake of ChPSf-ODN was five times as great as that of phosphorothioate oligonucleotides with 5'-fluorescein (PSf-ODN). However, nuclear uptake of ChPSf-ODN was only twice as great as that of PSf-ODN. The mechanisms of the different amounts of ChPSf-ODN distribution in the subcellular compartments are discussed. This study shows that the visual data derived from time-lapse confocal laser microscopy are helpful in understanding the cellular uptake mechanism of fluorescein labeled ODN.

抄録全体を表示

When primary cultures of bovine adrenal fasciculata cells were loaded with Calcium Green-1/AM, only 5～10% of cells were labeled. We have observed with fluorescence microscopic imaging that 1 pM adrenocorticotropin (ACTH) and 45 μM ATP induced a single giant Ca²⁺ spike followed by a plateau phase in around 3% and 80% of labeled cells, respectively. The ATP-responsive cells include the ACTH-responsive cells as a subgroup.
  Immuno- and histocytochemical staining revealed that Ca²⁺ signaling cells with ATP stimulation did not contain essential steroidogenic enzymes such as cytochromes P450scc, P450c21, and 3β-hydroxysteroid dehydrogenase. On the other hand, apparent ATP-insensitive cells were rich in these three enzymes. We assigned, therefore, ATP-insensitive cells as steroidogenic cells. Moreover, we have found that steroidogenic cells did not show Ca²⁺ signaling because of insufficient loading with Calcium Green-1/AM. It should be noted that with the presence of only 0.01% Triton X-100 we achieved sufficient labeling for more than 90% of total cells with Calcium Green-1/AM. In these preparations, 1 pM ACTH induced Ca²⁺ oscillations, step-like increase in Ca²⁺ and Ca²⁺ oscillations superimposed on a steplike increase for major population of cells (～90%) in addition to a single giant Ca²⁺ spike (Kimoto, Ohta and Kawato, 1996).
  In cell population measurements with a spectrofluorometer, the observed single Ca²⁺ spike would therefore be caused by non-steroidogenic cells upon hormonal stimulation, when cells are loaded with a Ca²⁺ indicator in the absence of Triton X-100.

抄録全体を表示

The pH change around an E. coli colony incubated on agar medium was visualized using a new type of pH imaging microscope, the scanning chemical microscope. This microscope utilizes a pH sensor which can work as a multiple array of sensing parts, and converts pH values into pH image. Changes in the pH distribution resulting from the metabolic activity of the E. coli colony were observed. Incubation was continued for more than 30 hours, and the temperature dependence of the metabolic activity was noted. This microscope is expected to be a powerful pH imaging tool for use in biology and chemistry.

抄録全体を表示

Matured Rhodomela confervoides is one of the marine algal species rich in bromine, while matured Gelidium amansii contains little bromine. Owing to the abundance of bromine in both young Rhodomela confervoides and Gelidium amansii, this particular element was mapped at tissue level using x-ray analytical microscope (Horiba Co.) for these two species. The results indicated that bromine was distributed almost evenly in Rhodomela confervoides while it concentrated strongly at the bud of the algal body in Gelidium amansii. These results demonstrate that halogen assimilation by bromoperoxidase and accumulation later in the algal body depend on two different systems, at least in some kinds of marine algae. It can be turned over quickly and not result in accumulation.

抄録全体を表示

Exocytotic secretion entails the cycles of fusion and removal of secretory granule membranes at the cell surface (exocytosis-removal cycle), occurring at proper sites and proper timing. Attempts were made to visualize these processes directly in living rat parotid acinar cells, by flooding the exocytosed granule space with fluid-phase fluorescent tracers (Lucifer yellow or FITC-dextrans) and by imaging its changes with confocal microscopy. With a delay of 10～15 seconds after addition of isoproterenol to stimulate secretion, exocytotic profiles, as indicated by the fluorescent spots, appeared along the luminal border abruptly in a granule-shape. Such profiles maintained their round shape for periods ranging from a few seconds to several tens of seconds, then gradually diminished and disappeared within a total time range of between 40 and 70 seconds. These images probably represent the spatio-temporal events involved in the exocytosis-removal cycle of single secretory granules, including stimulus-secretion coupling and exocytosis-endocytosis coupling, as indicated previously by biochemical and electrophysiological studies.

抄録全体を表示