RNase T₁ has two isozymes, Gln25- and Lys25-isoforms. The latter is relatively more stable than the former, although enzymatic activities are the same in both isozymes. Conformations of the two isoforms are not distinguished from each other crystallographically. To elucidate the mechanism of this phenomenon as based on the three-dimensional structure, energy minimization calculations with and without restraints were carried out for the complexes of guanosine 3'-monophosphate (3'-GMP) with Lys25-RNase T₁. The results indicated that the stability is mainly due to the electrostatic interaction of Lys25 with Asp29 and/or Glu31, not with Glu28 as reported previously.

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Practical imaging strategies are presented to investigate cell or structural systems which resemble Sierpinski carpet morphologies in biology. This class of patterns can be quantitatively measured with a Fractal Dimension to index structural complexity and scale-invariant system properties. Filamentous fungal colonies grow by repeated branching from the hyphal tips which develop into an interconnected (mycelial) network. This cell distribution is non-random and stochastically fractal. We investigate the regulation of hyphal branching by measuring the positional distribution of acid phosphatase, which is a marker enzyme for intracellular regions strongly implicated in coordinated regulation of the branching response. A nonequilibrium experimental growth environment acts to induce an adaptive response, and it is shown that the spatial distribution of this enzyme can be understood in terms of the Sierpinski fractal or a Percolation cluster. Percolation measures the ability of a fluid (e.g. enzyme) to spread or diffuse continuously. Image-analysis and data interpretation routines are developed to measure percolation properties arid this is related to cell morphology at different resolution scales to explore realistic cell development strategies.

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In order to develop a method to study secretion of insulin at a cellular level, we observed endocrine islet cells in the rat pancreas using a video-enhanced contrast differential interference contrast microscope. At a very high magnification (×10,000), islet cells were easily distinguished from acinar cells by their differences in appearance, and individual secretory granules of the islet cells were definitely resolved. When the concentration of glucose in the bathing medium was raised from 1.5 mM to 15 mM, many secretory granules showed abrupt light intensity changes and disappeared sequentially (degranulation). The cells that responded to glucose were immunopositive when fixed in mid-response and stained with anti-insulin antibody. These results indicate that the responses induced by glucose stimulation reflect the secretory activity of insulincontaining β-cells. Secretagogues of insulin, tolbutamide (100 μM) and A23187 (1 μM), induced even stronger responses than glucose. Direct visualization of these quantal responses in real time in pancreatic tissues will be very promising for studies of dynamics of insulin secretion.

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The preoperative planning for pelvic osteotomy was done on a stereolithography (SLA) model using our original algorithm, SurgiPlan, which could analyze images of three-dimensional computed tomography (3-DCT) and simulate various types of osteotomy. The subject cases were five women, of whom one had undergone Pemberton's operation; three, rotational acetabular osteotomy; and one, rotational acetabular osteotomy combined with advancement of the greater trochanter. Although the simulation with 3-DCT had advantages in processing the images for visualizing anatomy and lesions, its images remained two-dimensional. Stereolithography, on the other hand, was a real 3-D model which was able to give operators good anatomical orientation and a sense of security during surgery. Preoperative planning using a combination of 3-DCT and stereolithography had many cumbersome aspects, but this combined surgical navigation method was very useful and helpful in practical operations.

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We recently developed a new autofocusing method for chromosome aberration scoring which enables very fast scanning. This system utilizes, near infrared reflection from the surface of a glass microscope slide coated with an infrared reflector. In practice, another coating over the reflecting film is needed in order to protect the reflecting film from chemical agents. In the present study, the qualities of chromosome preparations on slides coated with SiO₂ or SiO over the reflecting film were compared visually and using the automated system. Chromosome preparations were made from cultured human lymphocytes and stained with Giemsa. Microscopic observation showed that stained cytoplasmic substances were found around chromosomes more frequently on SiO₂-coated slides than on SiO-coated slides. Metaphases were found, and their images were digitized at high magnification by an automated image analysis system. Cytogeneticists reviewed the digital images and counted false positives and metaphases unsuitable for scoring. “Misframed” metaphases, some of whose chromosomes were straddling the border of the monitor field due to errors of automatic centering or zooming, were observed more frequently on SiO₂-coated slides than on SiO-coated slides. The high rate of “misframed” metaphases was a major disadvantage for cytogenetic analysis by automatic facility of image grabbing. Most “misframed” metaphases were irregularly spread, suggesting the interference of SiO₂ coating with the circular (uniform) spreading of metaphases. Preparations on SiO-coated glass were superior in terms of the clarity of chromosome images, circular spreading of metaphases, and suitability for automated image analysis.

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Immunostaining and confocal laser microscopy were used to estimate relative amounts of murine attachmin in the macrophage plasma membranes at various periods of cell differentiation, i. e., M1, PU5-1.8, J774.1, TtT/M-87, peritoneal inflammatory macrophages, and resident macrophages. The results demonstrated that the various cells used in this study expressed attachmin in the cell membranes. However, the amount of attachmin was very different from one kind of cell to another. The strength of nonspecific adhesion of macrophages was almost proportional to the amount of attachmin in the cell membranes. It is suggested that the amount of attachmin in macrophages is available as a new differentiation marker for cells.

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