Takashi Makita

The effect of dimethyl sulfoxide (DMSO) in the incubation medium for the cytochemical localization of succinate dehydrogenase.

SummaryAddition of dimethyl sulfoxide (DMSO) to the cytochemical incubation medium for succinate dehydrogenase was attempted to accelerate penetration with consequent shortening of the incubation time. The copper-ferrocyanide medium for demonstration of succinate dehydrogenase activity was applied to fresh and hydroxyadipaldehyde-fixed muscle of the hamster and mouse and the albumen secreting gland cells of the hen oviduct. Cytochemical evidence indicated that DMSO did not seem to inhibit this enzymatic activity. With a shorter incubation time, less heterogeneity in reaction product was obtained in the mitochondria of muscle. The marked heterogeneity found in the reaction in the intracristal space of mitochondria also was diminished with addition of DMSO to the medium. The gland cells, whose ultrastructure was not well preserved with prolonged incubation, showed reductase reaction with the DMSO-containing medium.

IMMUNOCYTOCHEMICAL COLOCALIZATION OF DESMIN AND VIMENTIN IN HUMAN FETAL SKELETAL MUSCLE CELLS

Desmin and vimentin are the major intermediate filaments in muscle. They have been extensively studied in animal experiments. This study is the first to identify the distribution and to analyse the correlation of desmin and vimentin in human fetal skeletal muscle. Vimentin might be replaced by or transformed into desmin during myogenesis in chick embryo, although the precise process remains to be elucidated. The aim of this report is to evaluate the ratio of desmin to vimentin in human fetal muscle.

Calcium detection in secretion granules of avian oviduct by scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDX).

Secretion granules in the shell gland, isthmus, and albumin-secreting region of the hen oviduct were analyzed with WET-scanning electron microscopy (SEM) and EDX, a combination of wide-angle backscattered electron detector (BED) and energy-dispersive X-ray microanalyzer (EDX). Glutaraldehyde-fixed but unhydrated, unstained, and uncoated samples were analyzed; Ca was localized in all secretion granules in all three sections of the hen oviduct studied.

Inhibition of peroxisomal degradation by 3‐methyladenine (3MA) in primary cultures of rat hepatocytes

A significant reduction in peroxisomes has been demonstrated in primary cultures of rat hepatocytes. This report demonstrates that 3‐methyladenine (3MA), a potent inhibitor of autophagy, inhibits this effect.

Molecular organization of hepatocyte peroxisomes.

The matrix of peroxisomes has been considered to be homogeneous. However, a fine network of tubules is visible in electron micrographs at very high magnification. This substructure becomes more positive in a high-contrast photocopy and with an imaging-plate method. Clofibrate, bezafibrate, and aspirin increase peroxisomes. In proliferated peroxisomes, the density of matrix is low and the fine network is more visible. The effect of proliferators is more significant in males than in females. This sex difference may involve the action of estrogen, growth hormone, cytochrome P-450 and thyroxine. Mg-ATPase is localized on the limiting membrane of peroxisomes. Even on the membrane of irregular projections of proliferated peroxisomes, Mg-ATPase is evident cytochemically. Carnitine acetyltransferase is detectable in the matrix of proliferated peroxisomes. Withdrawal of proliferators results in a rapid decrease of peroxisomes. This may indicate the existence of peroxisome suppressors. Alternatively, dynamic transformation of vesicular to tubular types in peroxisome reticulum may occur. Such transformation has been described in lysosomes and mitochondria.

Age-related changes in the susceptibility to clofibric acid, a hypolipidemic agent, of male rat liver.

Age-related changes in the susceptibility to clofibric acid were investigated in male F344 rats of 8, 52, and 117 weeks old. Hepatomegaly, decrease of serum total cholesterol and triglyceride, increase of the total cytochrome P-450 contents, induction of the activities of microsomal omega-hydroxylation and peroxisomal beta-oxidation, proliferation of smooth endoplasmic reticulum and peroxisomes were detected in 8- and 52-week-old rats. In 117-week-old rats clofibric acid treatment resulted in decrease of serum total cholesterol, elevation of the activities of microsomal and peroxisomal enzymes, and slight proliferation of peroxisomes. These results suggest that the susceptibility of the male F344 rat liver to clofibric acid decreases in 117-week-old rats, though the effect is still recognizable.

Redistribution and fate of colchicine-induced alkaline phosphatase in rat hepatocytes: possible formation of autophagosomes whose membrane is derived from excess plasma membrane.

The redistribution and fate of colchicine-induced alkaline phosphatase (ALPase) in rat hepatocytes were investigated by electron microscopic enzyme cytochemistry and biochemistry. ALPase activity markedly increased in rat hepatocytes after colchicine treatment (2.0 mg/kg body weight, intraperitoneal injection). At 20–24 h after colchicine treatment, the liver showed the highest activity of ALPase. Thereafter, ALPase activity decreased and returned to normal levels at 48 h. In normal hepatocytes from control rats, ALPase activity was seen only on the bile canalicular membrane. However, at 20–24 h after colchicine treatment, colchicine-induced ALPase was redistributed in the sinusoidal and lateral (basolateral) membranes as well as in the bile canalicular membrane. At 30–36 h after colchicine treatment, ALPase activity on the basolateral membrane gradually decreased. In contrast, ALPase in the bile canalicular membrane increased along with the enlargement of bile canaliculi, suggesting that ALPase in the basolateral membrane had been transported to the bile canalicular membrane. Furthermore, ALPase-positive vesicles, cisternae and autophagosome-like structures were frequently seen in the cytoplasm. ALPase was also positive in some lysosomal membranes. ALPase in hepatocytes at 48 h after colchicine treatment returned to almost the same location as in control hepatocytes. Altogether, it is suggested that excessively induced ALPase is at least partially retrieved by invagination of the bile canalicular membrane and then transported to lysosomes for degradation. In addition, this study indicates that excess plasma membrane might be a possible origin of autophagosomal membrane.

X-ray microanalysis and electron microscopy of platinum complex in the epithelium of proximal renal tubules of the cisplatin administered rabbits.

The initial phase of endocytosis of cisplatin, an anti-tumor platinum agent, into epithelial cells of the proximal renal tubules of rabbits was studied using an X-ray microanalyser at the electron microscopic level. After one to 11 intravenous injections of cisplatin (1 mg/kg/daily), each rabbit was sacrificed with overdose of pentobarbital and small pieces of renal cortex were fixed with 4% glutaraldehyde and 2% osmium tetroxide. After binding on the surface of brush border, dense substance was taken up in endocytic vacuoles that proceeded into the center of epithelial cells leaving empty or scanty vacuoles in apical area. Both platinum and iron were detected in such intracellular dense substance. This shows the transcellular pathways of platinum complex. On the other hand, intercellular pathways were not found in this experiment.

Immunocytochemistry of Apoptosis Induced by Bromodeoxyuridine in Human Leukemic HL‐60 Cells

Various antitumor drugs which target tumor cells in the S‐phase of the cell cycle induce apoptosis in HL‐60 cells. The present study shows that apoptosis is inducible in HL‐60 cells by continuous exposure to bromodeoxyuridine (BrdU), an S‐phase specific reagent and a thymidine analogue. The localization of BrdU in the apoptotic cells was visualized with immunogold particles using an antl‐BrdU monoclonal antibody. After 3 days of treatment with 20 μM BrdU, cells ceased to grow and their viability was significantly decreased. Morphologically, nuclear segmentation and cytoplasmic maturation were observed in many viable cells, and the dead cells showed typical features of apoptosis. The nuclear DNA of apoptotic cells was visualized by the immunocytochemical detection of BrdU with post‐embedding immunogold staining. The condensed chromatin of the apoptotic cells was highly labeled with immunogold particles, while the nucleolus was sparsely labeled. The chromatin could be traced with BrdU‐immunocytochemistry even after disappearance of the nuclear envelope. These results indicated that apoptosis in HL‐60 cells was induced by incorporation of BrdU into DNA of the S‐phase cells. Using BrdU‐immunocytochemistry, the localization of nuclear DNA in apoptotic cells was visualized and the changes of nuclear structures were followed during the progression of apoptosis.

Scanning electron microscopy and cytochemical localization of carnitine acetyltransferase activity in normal and dystrophic muscle of mice

SynopsisA scanning electron microscopic survey of normal and dystrophic murine muscle revealed an increased amount of fat around the dystrophic muscle. Although decreased activity of carnitine acetyltransferase an enzyme involved in lipid metabolism, has been reported previously in dystrophic muscle, it was found in this study that the electron microscopic localization of this enzyme in dystrophic and normal muscle was similar. The final reaction product for this enzyme, uranyl ferrocyanide, was located on the outer surface of the inner membrane of mitochondria.Although not strictly specific to the dystrophy, some focal alterations of the submitochondrial structure, such as an intracristal dense line and the whorl-like arrangement of cristae and an increased number of lipid droplets closely associated with mitochondria, were conspicuous in the affected muscle fibres. No carnitine acetyltransferase activity was detected on these altered structures.