A novel method was developed to estimate the relative amounts of total and local attachmin in immunostained mouse peritoneal macrophages using confocal laser microscopy. Macrophages were made to adhere to an uncoated glass surface; attachmin in the cells was immunostained with a polyclonal anti-attachmin antibody and a FITC-labeled secondary antibody after treatment with or without Nonidet P-40. Fluorescence in thin optical sections through the vertical and tangential levels of the glass surface was quantitatively measured with a confocal laser microscope; relative amounts of total and local attachmin per cell were estimated during the cell adhesion to glass. Using this method revealed that the attachmin was distributed all over the macrophage plasma membranes homogeneously; at the free surface of the cell, approximately half of the ≥ 300-kDa attachmin polymer was found to protrude outside the cell through the plasma membranes. This simple method will be used for quantitative analysis of the distribution and localization of various insoluble antigenic components within cells.

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We tried to obtain ratio-images of Ca²⁺ responses in cultured rat hepatocytes using indo-1 and newly developed video-rate ultraviolet (UV) laser-scanning confocal microscope systems. Indo-1 was excited by 351 nm laser light, and the emissions at 400—440 nm and over 440 nm were measured. To get precise ratio-images the systems were adjusted to correct chromatic aberrations in the UV range. As a result, the focal points and the depths of field became almost the same at both emission wavelengths. The ratio-images were usually made by averaging eight successive frames to improve the signal-to-noise ratio at 3.75 images/sec. The in situ calibration yielded the basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 0.1 μM in nuclear regions and that of 0.2 μM in cytoplasmic regions. Phenylephrine (15 μM) elicited Ca²⁺ oscillations. The increase in [Ca²⁺]_i in each oscillation originated from a specific region near the plasma membrane and propagated to the other side of the cell in 2—4 sec. [Ca²⁺]_i in the nuclear regions also increased to about the same levels as in the cytoplasmic regions. [Ca²⁺]_i then decreased slowly in 10—20 sec. The patterns of Ca²⁺ waves were always the same in the cells studied.

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We have developed a simple method to predict the effect of mutations of bases and amino acid residues on the binding affinity between protein and DNA, automated the procedure and visualized the results on computer graphics system. This method evaluates mostly local effects such as steric hindrances, H-bonds, and van der Waals interactions, which reflect a rough measure of surface complementarity in the interface of interacting molecules. We discuss its application to the interaction between λ repressor and DNA. Despite its simplicity, the results of the computer simulation have shown a high correlation with experimental results. Therefore, the method can be effectively used for investigating the mechanism of protein-DNA recognition, and serves as a useful guide for designing mutagenesis experiments.

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The 3D distribution of membranous organelles in living cultured cells was visualized by confocal laser scanning microscopy combined with lipophilic probe staining. When cultured 3Y1 fibroblasts were stained with a fluorescent dicarbocyanine, DiOC₆(3), mitochondria, endoplasmic reticulum and plasmalemma were visualized at high resolution under a confocal laser scanning microscope. Stereo pair images reconstructed from serial optical sections of such stained cells clearly showed the 3D distribution of mitochondria and endoplasmic reticulum throughout the cytoplasm. Microvilli were also seen to protrude from the cell surface. BODIPY-conjugated ceramide selectively accumulated in the Golgi apparatus to provide sharp 3D images of the organelle. For counter-staining of such stained living cells, we used dihydroethidium which selectively stained nuclei and chromosomes. We conclude that confocal 3D microscopy combined with lipophilic probe staining is a useful approach for better understanding not only of the 3D distribution but also of the dynamic features of membranous organelles in living cells.

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We have shown that the population of receptors for uPA of a human metastatic carcinomatous line, Detroit 562, becomes homogeneous, having a single affinity by the selection of one clone (C5) (Takahashi et al., 1991). To study the activities of uPA associated with this cloned cell, a method for determination was developed by use of a nitrocellulose membrane filter to which colonies grown on a culture dish were transferred. The filter was found to be adequate for the analysis of uPA activity at the cellular level by zymography, and it was possible to carry out an immunochemical assay by incubation with antibody against uPA. The determination of the lytic areas (plaques) developed on the zymogram was carried out by a computer-imaging method (Takahashi et al., 1993). It was found that the plaque size was linearly proportional to the uPA activity, suggesting that the increment of the plaque followed a process of plasmin diffusion in the zymographic gel matrix under the experimental conditions using purified uPA immobilized on the filter. Thus, the method enabled us to analyze a large number of colonies, and made possible the construction of a two-dimensional map plotting colony- versus plaque-size, demonstrating that the cell population was heterogeneous, consisting of subpopulations of cells having diferent levels of active uPA: higher, moderate, lower, and no expressed level. Because of an apparent homogeneity in the amount of uPA antigen associated with the colonies, those levels might be due to a locally restricted autocrine modulation by certain inhibitors produced by colonies.

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A simple system for the semi-automatic detection of numerical abnormalities appearing in chromosomes in a tumor was developed. The system consists of a fluorescence microscope, a personal computer, and a microscope stage which can be controlled three-dimensionally by the computer. A DNA probe of a specific sequence was hybridized on chromosomes in cells of interphase, and the probe was labelled with fluorescein isothiocyanate (FITC). The ratio of the number of nuclei in which the number of hybridized parts per nucleus was ≥ 3 to the total number of nuclei was obtained semi-automatically by the computer system. The results obtained by this system were compared with those by the human eye for the same preparation were compared. The difference between the two methods was less than 5%, demonstrating sufficient accuracy for practical use.

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A program is described that deals with a frame image in protein crystallography. This program is developed in order to display the diffraction image, to integrate the pixel data, and to reduce to structure factors obtained by use of the Weissenberg camera installed in Photon Factory. Use has been made of the library of X-Window and X-Toolkit for the man-machine interface parts in the program, which makes it highly interactive. The program has been written in the C language for UNIX machines and is portable to almost all kinds of graphics workstations.

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On the basis of chemical modification and X-ray crystallography, glutamic acid at position 58 (Glu-58) of ribonuclease T₁ (RNase T₁) has been identified as a catalytic residue. On the other hand, the mutant of RNase T₁ in which Glu-58 is replaced with aspartic acid (Glu58Asp RNase T₁) or alanine (Glu58Ala RNase T₁) did not lose the enzymatic activity completely. To elucidate the mechanism of this phenomenon as based on the three-dimensional structure, molecular dynamics simulation and energy minimization calculation were carried out for the complexes of guanosine 3'-monophosphate (3'-GMP) with Glu58Asp and Glu58Ala RNase T₁. The conformation thus obtained was compared with that of the complex of 3'-GMP and wild-type RNase T₁. The results indicated that upon replacement of Glu-58 with Asp or Ala, the relative position of His-40 to 3'-GMP in the active site change in such a way that His-40 acts as a general base catalyst.

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The organization of cytoskeletal components in early Drosophila melanogaster embryos during cellularization was examined by fluorescence microscopy including confocal laser microscopy. In the embryo, at late telophase of the last (fourth) nuclear division of the syncytial blastoderm stage, confocal lasar microscopy revealed that several bundles of astral microtubules run from the poles to the surface plasma membrane, where a new cleavage furrow was expected to form. When the cleavage furrow began to proceed between the cortical nuclei, microtubules were rearranged into a parallel bundle to surround each elongating nucleus, extending from the apical centrosomes. Additionally an intense staining of actin and myosin was observed at the tip of the cleavage furrow, forming a prominent actin ring. Alpha-actinin was co-localized with both actin and myosin along the cleavage furrow on the plasma membrane, especially at its advancing tip. The distal ends of such microtubules often were spatially associated with the actin ring. Subsequently, the actin ring started to constrict the cleavage furrow below the nucleus to separate each cell from the central yolk region. Some microtubules extended down to the yolk region and remained in the cytoplasmic strand bridging a newly formed cell to the central yolk region after the completion of cellularization. These observations suggest that both microtubules and actin-myosin systems may play crucial roles in the formation of cleavage furrows in a cooperative manner during cellularization of Drosophila melanogaster embryos.

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We developed a three-dimensional (3-D) imaging apparatus for assessing preoperative status and surgical results, using a 3-D solid modelling system with resin which is polymerized by exposure to ultraviolet (UV) laser beam. 3-D data which are constructed with multiple consecutive 2-D echograms were used for the system. The 3-D data were collected with ordinary echography and processed in combination with a previously developed 3-D digitalizer. Using the system, a resin model of the vasculature of the hepatic vein was reconstructed with real shape within about 5 hours of processing time. As the reconstructed plastic model can be observed at any time from any direction, it can be used for surgical simulation. Compared with another 3-D display unit without the solid model, the present method for reconstructing an image as a real resin model is more advantageous for the preoperative simulation of proposed surgery and provides the surgeon with a precise preoperative assessment of surgical intervention.

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