Masataka Morita

Biological micro-particles in rain water

This work constitutes the first report on the morphology and chemical identification of biological micro-particles in rain water at two sites (Shizuoka and Tsukuba) from Japan. Micro-particles in rain water were deposited directly onto electron microscopic grids using ultracentrifugation. Particles with diameters more than 0.2 µm were examined using an electron microscope equipped with an energy-dispersive X-ray analyzer. Biological particles in rain water were found to be bacteria and leaf debris, and most of those particles were associated with minerals. Peaks of phosphorus and potasium in the X-ray spectra, which are characterized as chemical components of bacteria or leaves, were observed in all the biological particles. These particles in rain water were present in number percentages of 23 to 34% of the total insoluble particles, with concentrations of 1.6×104 to 2.4×104 cm−3 of rain water. This finding suggests that these biological particles are captured effectively in cloud water and/or precipitation, and that they may play a role in cloud condensation and/or ice nucleation.

One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles

In the present study, we describe a new procedure using freezing‐thawing to density gradient solution of Nycodenz for one‐step separation of organelles from the rat liver and subsequent proteome analysis of subcellular fractions. To prepare two‐dimensional electrophoresis (2‐D PAGE) profiles of tissue organelles, we performed one‐step subcellular fractionation of rat liver homogenate using a density gradient of Nycodenz solution, which resulted in the separation of the cytosolic fraction from the postnuclear supernatant. The density gradient of Nycodenz was prepared from a 20% solution in a centrifuge tube by freezing‐thawing overnight at –20°C and at room temperature for a few hours without the initial centrifugation procedure. The shape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined using one‐dimensional sodium dodecyl sulfate (SDS)‐PAGE. The organelles were confirmed using Western blotting. Our results indicate that our procedure provides a simple method for the separation of organelle fractions from the rat liver tissue.

A simple semisolid subtraction method using carbodiimide-coated microplates

In this article, we develop a novel subtraction method using carbodiimide-bound microplates. This method utilizes the high affinity of carbodiimides for both single- and double-stranded nucleic acids. Carbodiimide-mediated end-attachment of driver RNA to microplates allows semisolid phase hybridization between driver RNA and target cDNA, and ensures easy removal of RNA/cDNA hybrids composed of the genes commonly expressed in driver and target. As a result, the target-specific genes are left unhybridized and enriched in the hybridization supernatant. We define the optimal conditions for the method as a target/driver RNA ratio of 1:10 and a period of hybridization of 24 h. There are at least three major advantages with the present method: (1) The entire procedure, which consists of two steps, is very simple; (2) hybridization efficiency can be monitored before further processing of the samples; and (3) rare transcripts can be effectively enriched. This method may be a powerful tool to isolate the genes specifically expressed in particular cell or tissue types, and is easily applicable to many studies in molecular biology and genetics. Isolation of polyploid megakaryocyte-specific genes is shown as an example.