A monovalent cation-sensitive actin-binding factor in a myeloid leukemia cell line

Cell extracts of a murine leukemia cell line, M1, apparently contain three kinds of actin-gelation factors; a filamin-like protein, and 38K-dimer and 105K-dimer proteins. Unlike gelation by the filamin-like protein, gelation by the latter two proteins is inhibited by low concentrations of KCl. Our study of the 38K protein has been reported elsewhere (Takagi, K. et al., J. Biochem. Tokyo 97, 605-616, 1985). We here describe the purification and characterization of the 105K protein. The 105K protein differs from the alpha-actinin group of proteins in its antigenicity, peptide components and Ca2+-insensitivity. The saturated binding ratio of the protein to purified skeletal muscle actin is 1:8; when this ratio exceeds 1:20, gelation takes place. This gelation is inhibited completely by the presence of 25 mM KCl. Electron microscopy revealed that, in the absence of KCl, the 105K protein/actin mixture forms short actin bundles that are accompanied by a meshwork of short single filaments. The presence of 25 mM KCl did not prevent actin-bundling, but the bundles became longer and the meshwork of short filaments was no longer present.     

Distribution of Leu 7 (HNK-1) antigen in human digestive organs: an immunohistochemical study with monoclonal antibody

Summary Leu 7 immunoreactivity was demonstrated with the indirect peroxidase-labelled antibody method on frozen and paraffin-embedded tissue sections of human digestive organs. Anti-Leu 7 monoclonal antibody, which allegedly detects mononuclear cells with natural killer or killer activity, recognized lymphoid cells among intestinal epithelial cells and in the germinal centres of solitary lymphoid follicles of small and large intestine, and a few in gallbladder, liver and the lamina propria of the intestine. In addition, peripheral nerve fibres, endocrine cells in the gut and pancreas and carcinoid and islet cell tumours were also positively stained. At the ultrastructural level, Leu 7 antigen was localized on the plasma membrane of granulated lymphoid cells in the gut mucosa and on the secretory granules of intestinal endocrine cells. In normal pancreas, Leu 7 immunoreactivity was demonstrated in most cells containing pancreatic polypeptide and in many cells containing somatostatin or glicentin. Insulin-containing cells, however, lacked Leu 7 immunoreactant. These findings were obtained in both frozen sections and paraffin-embedded sections. The possible cross-reactivities of monoclonal antibodies are discussed as they raise an important caveat in immunohistochemical studies using these antibodies.     

Quantitative fluorescence microscopy on dynamic changes of plastid nucleoids during wheat development

Summary Dynamic change of plastid nucleoids (pt nucleoids) was followed by fluorescence microscopy after staining with 46-diamidino-2-phenyl indole (DAPI). The fluorescence image was quantified with a supersensitive photonic microscope system based on photon counting and image analysis. The results showed that small pt nucleoids located in the center of proplastids in the dry seed increased in size after imbibition and formed highly organized ring structures in the dark, which divided into ca. 10 pieces within 3 days. Corresponding to this morphological change, DNA content of a plastid multiplied 7.5 fold. Total increase in DNA content of pt nucleoids per cell was 34 times as that of dry seed, as plastid multiplied 4.6 times in the average during this period. Upon light illumination small pt nucleoids having basic genome size were separated from divided pt nucleoids, suggesting a relationship with the formation of thylakoid system. The significance of the procedure established in this study is discussed in analysing the dynamic changes of intracellular small genomes.On leave from Department of Biology, Faculty of Science, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.     

Histochemical studies on the regeneration of aminergic nerves in rat cerebral artery after superior cervical ganglionectomy

The course of regeneration of aminergic nerves in rat cerebral arteries was studied by means of histochemical methods, after uni- or bilateral cervical sympathectomy. Degeneration of aminergic nerves started on day 1 and was complete between days 3 and 7 after surgery. Between weeks 4 and 6, regenerating nerves started to appear from the proximal internal carotid artery. Regenerated aminergic nerve fibres were generally unbeaded and intensity of fluorescence was weak. The circular nerves appeared earlier than the longitudinal ones. The number of regenerating nerves reached the maximum, between months 9 and 12, at about half the normal level. AChE activity of the cerebral arteries showed no significant changes at any stage.     

Incorporation of tritiated thymidine into mitochondrial DNA of the liver and kidney cells of chickens and mice in tissue culture

Summary ³H-thymidine incorporation into mitochondrial DNA of the liver and the kidney cells of chick embryos and newborn mice in tissue culture was shown by means of electron microscope radioautography with accurate localization. In these cells, about 20% of all the mitochondria were labeled at their matrices between the cristae within 4 hours in contact with the radioisotope, which were removed by DN'ase.From the results, it is clear that the mitochondria of avian and mammalian cells in tissue culture synthesize DNA.     

Effects of serine protease inhibitors on oxyradical burst from phagocytizing neutrophils—analysis by chemiluminescence counting and its microscopic imaging

Reaction difference of oxyradical generation and luminol-dependent photoemission of zymosan- and phorbol ester-treated neutrophils were investigated using a conventional photomultiplier and ultrasensitive photonic imaging technique. Zymosan-treated cells released a concentrated photonic burst corresponding to the cellular distribution. In contrast, phorbol ester-treated cells produced a negligible level of photoemission, and the additional application of Ca²⁺ ionophore enhanced the photonic burst, which was gradually spread out into extracellular space. Serine protease inhibitors did not attenuate PMA-induced chemiluminescence but did attenuate zymosan-induced chemiluminescence. This suggests the involvement of serine protease in the respiratory burst of phagocytizing neutrophils.     

Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.     

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.