Akira Yamamoto

Accurate determination of DNA content in single cell nuclei stained with Hoechst 33258 fluorochrome at high salt concentration.

SummaryIn an attempt to achieve accurate quantification of DNA levels in cell nuclie, we studied the influence of salt concentration on the fluorescence of cell nuclei complexed with Hoechst-33258 (Hoe) fluorochrome. The fluorescence of cell nuclei was compared with that of extracted DNA as well as that of nucleosome core. Conformational changes in these complexes were examined by measuring both fluorescence anisotropy and fluorescence lifetime in the nanosecond region. The results showed that the fluorescence of DNA-Hoe was quenched by the nucleosomal structure, there being an associated increase in anisotropy and a decrease in the fluorescence lifetime; however, the fluorescence was restored to the orginal level by the addition of a high concentration of NaCl, CsCl, or LiCl. The reduction in fluorescence may have been due to loss of fluorescence energy caused by collision of the fluorophore with histones in the nucleosome. The addition of 1 M NaCl to the medium used for staining with Hoe greatly stabilized the fluorescence of DNA in cell nuclei. The DNA content of individual cell nuclei was determined by comparing the fluorescence of these nuclei with that of a standard DNA solution. For lymphocytes and liver ploidy cells, reasonably accurate values were obtained by applying the present method.

Fatigue fracture of the ulna occurring in pitchers of fast-pitch softball

We have reported three cases of fatigue fracture of the ulna in male pitchers of fast-pitch softball. To elucidate the etiology of injury, we first selected three healthy male and three healthy female pitchers from a well- trained college team and analyzed their forearm move ment by high-speed cinematography. This showed slight flexion of the elbow joints during wind-up motion, dorsal flexion of the hand joints upon releasing the ball, and extreme pronation of the forearms during the fol low-through. We then took 8 mm CT scanning sections of the forearms. Using these images, we investigated shapes and areas of cross-sections of the ulna and its cortical and cancellous bones from the elbow to the hand joints. Our results reveal that the shapes of the sections are significantly different from circles at around the center of the ulna, and the cross-sectional areas are smaller in the middle one-third of the ulna than in other parts. These observations imply that fatigue frac tures of the ulna in pitchers of fast-pitch softball must be torsionally induced, tending to occur at the middle one-third of the bone.

Effect of radical retropubic prostatectomy on erectile function, evaluated before and after surgery using colour Doppler ultrasonography and nocturnal penile tumescence monitoring

Objective To assess the effect of radical retropubic prostatectomy on erectile function, by evaluating objectively patients erectile function before and after surgery.

Feasibility of multi‐slice computed tomography in the diagnosis of arteriogenic erectile dysfunction

Objective To compare computed tomography (CT) angiography (CTA) obtained by multi‐slice CT (a new minimally invasive method) with the current standard of arterial imaging, digital subtraction angiography (DSA), in diagnosing arteriogenic erectile dysfunction (ED).

Three-dimensional CT cavernosography: reconsidering venous ligation surgery on the basis of the modern technology

Study Type – Therapy (case series) u2028Level of Evidenceu20034

Pineal microdialysis in freely moving rats

We describe a surgical technique to implant the guide cannula for in vivo microdialysis in the rat pineal gland. This technique has the following features and advantages: (a) does not require ligation of the superior or transverse sinus, (b) minimizes bleeding from the dural veins, (c) does not disturb the sympathetic innervation originating from superior cervical ganglia, which is essential for pineal function. This new technique makes it possible to carry out chronic pineal microdialysis of freely moving rats.

NaCl-aided Hoechst 33258 staining method for DNA quantification and its application

SummaryWe investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome.The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.

Comparison of postmortem autolysis in cardiac and skeletal muscle.

To understand the mechanism in postmortem autolysis better, processes in the postmortem degradation of myofibril proteins in the presence of protease inhibitors were studied. Male Wistar rats were given injections of the carboxyl-, thiol-, and serine-protease inhibitors, pepstatin, Ep-475[L-transepoxysuccinyl-leucylamide(3-methyl) butane; E-64-C], and chymostatin, via the femoral vein. Control rats were similarly treated with saline. Then, myofibril proteins were isolated from their cardiac and femoral muscles and from those of control animals at various times after death, and degradation of these myofibril proteins with time was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In cardiac muscle, alpha-actinin was degraded rapidly, followed by the heavy chain of myosin and light chain of myosin (L2). Actin and the light chain of myosin (L1) were degraded slowly. the degradations of the heavy chain of myosin, alpha-actinin, tropomyosin and L2 after 14 days were not inhibited by pepstatin, but were inhibited by Ep-475 and chymostatin. In skeletal muscle, L1 and L2 were degraded rapidly, followed by the heavy chain of myosin and alpha-actinin. Actin was degraded slowly and was still unchanged 2 weeks after death. The degradations of protein components were inhibited by pepstatin, Ep-475 and chymostatin. These results indicated that after death the components of myofibrils are degraded by various proteases at various rates depending on their properties or structures. This degradation is fundamentally the same in cardiac and skeletal muscles, but inhibitors have somewhat different effects on the postmortem degradation processes after death in the two types of muscle.

Immunocytochemical localization of prostaglandin endoperoxide synthase in the bovine intestine.

The localization of prostaglandin (PG) endoperoxide synthase in bovine intestine was examined immunocytochemically with polyclonal antibody raised against PG endoperoxide synthase purified from bovine seminal glands. The most intense positive staining reaction for the enzyme was present in mast cells. Mast cells were found to be widely distributed in the intestinal wall, and were particularly numerous in the lamina propria. Most of the mast cells in the lamina propria of the intestinal villi were elongated and oriented with their long axis parallel to the plane of the absorptive epithelium. In whole mount preparations of jejunal villi, mast cells were seen to form a two-dimensional network in the lamina propria. In addition to mast cells, smooth muscle cells of the inner circular muscle layer and muscularis mucosae, nerve cells and fibers, endothelial cells of arterioles, and serosal epithelial cells also showed faint to moderate staining for the enzyme. These results suggested that mast cells are the major source of PGs in the bovine intestinal wall. The characteristic arrangement of mast cells in the intestinal villi may be related to their functions in this portion of the bovine intestine.

Virtual cavernoscopy: a novel diagnostic tool for use in the corpus cavernosal lumen in patients with erectile dysfunction

Study Type – Diagnostic (case series)