Ken Fujimori

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.

Trace elements found to be variable in two coral reef species,Heliofungia actiniformis andGalaxea fascicularis, collected from the ryukyu islands

Biominerals and metals of intertidal corals of two species (Heliofungia actiniformis, Quoy and Gaimard;Galaxea fascicularis, Linnaeus), collected from the Iriomote Island of Ryukyu, were examined with an inductively coupled plasma atomic emission spectrometer (ICP-AES). Twelve elements were detectable in the coralline skeletons dissected radially along the growth axis. The relative content (RC) of Hg periodically fluctuated and was minimum at the hollow sites of the coralline slab ofHeliofungia sp., corresponding to the cyclic growth. There were two types of elements: constant elements and variable elements along the growth axis. RCs of Ca, Mg, A1, Si, and P were nearly constant. RCs of Fe, Mn, Cu, and Ba were variable, but not as regularly changed as Hg. There were positive mass correlations of Hg to Mn, Cu and Zn, but not to Ba and Fe. In contrast, these relationships were not prominent and were likely degraded by aging in the skeleton ofGalaxea sp., suggesting a different mode from that of theHeliofungia sp.

Association of neutral α-glucosidase with cytoplasmic granules of peritoneal macrophages☆

Abstract A neutral α-glucosidase (EC 3.2.1.20) activity was shown to be associated with granules which are sedimentable at 10 000 g after differential centrifugation of mouse peritoneal macrophage homogenates. When the post-nuclear supernatant was centrifuged in a sucrose density gradient, high activities for neutral α-glucosidase and β-glucuronidase (EC 3.2.1.31) were detected in the bottom fractions because of aggregation of the granules. Neutral α-glucosidase-containing granules were completely disaggregated by the addition of 20 units/ml of heparin and 10 mM Tris-HCl (pH 7.2), which caused only a partial disaggregation of β-glucuronidase-containing granules. The addition of a high concentration of heparin, Tris buffer, or KCl to the gradient gave the same patterns of disaggregation of the granules. Under the condition in which about 50% of the total β-glucuronidase activity was released into the medium, depending on phagocytosis, very little α-glucosidase was released. These observations suggested that neutral α-glucosidase may localize in non-lysosomal granules.

Trace metals in vertebral columns of deep-sea teleost fish

Deep-sea teleost fish were collected from the Sagami Bay near a deep fissure in the Pacific Ocean. Fish were identified as Chlorophthalmis albatrosis, Engyprosopan xystrias, Satyrichthys hians, Ventrifossa garmani, and Halieutaea stellata. The Etmopterus lucifer is not a teleost, but a deepsea shark. Just after being caught and fixed in neutral 20% formol, the vertebral column was resected and prepared for measurement by inductively coupled plasma-atomic emission spectrometry.Trace elements were found to be Al, Si, Ti, Fe, Cu, Cd, Zn, and Hg at micrograms per gram levels. Major elements were Mg, Ca, P, and S at the milligram per gram level. Some of trace elements, Zn and Hg, were also usually found at this level.

A special comment on trace metals in the viscera of Turbo marmoratus, a toxic snail, on Ryukyu Island

Trace metals in the viscera of Turbo marmoratus, a huge conical spire, in the coral reef of Ryukyu Island were analyzed. The highest amount of Zn and Fe was detectable in the midgut gland known to be polluted by saxitoxin. This intestine maintained a considerable amount of Si (higher than Ca) which fluctuated in parallel with the taurine amount. These data suggest that the viscera of the alimentary tract accumulate some metals, especially Zn and Fe.