The localization of guanylate cyclase activity was studied cytochemically in the rod outer segments of rats light and dark-adapted for various intervals. A brown colored reaction was detected in rod outer segments under a light microscope. Under an electron microscope, a significant change in the distribution of the reaction products under light and dark adaptation was observed. When rats were dark-adapted for longer than 90min, an intense activity was found on the cytoplasmic side of the plasma membrane, and most of the disc membranes showed lower activity than in light with the exception of a few disc membranes near the pigment epithelium which showed rather intense activity. At 1sec illumination, no significant change was found in the distribution of the guanylate cyclase. The first significant change was detected at 5sec of illumination. After a 15sec light exposure, the reaction products of guanylate cyclase activity were observed on all the disc membranes regularly, and the activity on the plasma membrane was very weak. The present results indicate that light affects the guanylate cyclase activity and its distribution in the rod outer segments, suggesting that guanylate cyclase or cyclic GMP plays an important role in the control of ion permeability in the rod outer segment plasma membrane and a possible role in the visual adaptation process.

View full abstract

It is well known that mammalian spermatozoa utilize fructose as an energy source, and glycerol can also be metabolized for this purpose. Further, oxidoreductases related to energy metabolism, such as sorbitol dehydrogenase and α-glycerophosphate dehydrogenase, are contained in the testis. These enzymes contribute fructose synthesis and glycerol metabolism, respectively.
In this paper, the precise localization of these energy metabolism-related oxidoreductases was studied by electron microscopy. The results indicated that sorbitol dehydrogenase located in the cytoplasm of Sertoli cells adjacent to the late stage spermatids. On the other hand, α-glycerophosphate dehydrogenase was recognized in the mitochondria of late stage spermatids and spermatozoa. Therefore, fructose is presumably synthesized in the cytoplasm of the Sertoli cells, and glycerol is metabolized at the mitochondria of the sperm.

View full abstract

Various fixatives including acetone, ethanol, formalin, paraformaldehyde with picric acid, and glutaraldehyde were used for the staining of alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGTP) activities in human fresh frozen liver sections by an azo-coupling method. The staining intensity was variable among the fixatives; however, acetone gave best preservation of both enzyme activities.

View full abstract

Effects of DEHP (di-2-ethylhexyl phthalate) were studied on renal tubular epithelial cells of rats. The 1%, 2% and 4% (w/w) DEHP administered and control rats were sacrificed at 1, 2 and 4 weeks. The tissues were incubated in alkaline DAB medium and embedded in Epon. The 0.5μm thick sections were observed by light microscopy with an ocular micrometer. The number of peroxisomes was determined for the transected area per 25μm² in 40 different tubular cytoplasms. The differences in the number of peroxisomes between the experimental and control groups were evaluated from the viewpoint of various responses depending on the duration of administration, dose of DEHP, parts off the nephron and sex. Peroxisomal proliferation in DEHP-treated rats occured at low levels of dosage in a short time. The differences in increase between straight parts and convoluted parts of proximal tubules became the most obvious over a long time. The difference depending on sex was not so apparent. The response of proximal tubular epithelial cells to DEHP is not similar to that induced in liver cells by the same drug. Peroxisomes in renal tubular epithelial cells may play a role in lipid metabolism to a lesser extent than those in liver cells.

View full abstract

A concurrent microchemical and topochemical study of the tetrazolium reduction by isolated chloroplasts of Vicia faba under illumination was carried out. The microchemical analysis with INT^* revealed that the photosystem responsible for the reduction was the PS-II, because the addition of DCMU, DCPA, MCC, CIPC or CAT to the reaction medium at low concentrations markedly suppressed the formazan production. The topochemical analysis with BSPT, combined with the densitometric one, revealed that the electron transfer activity mediated by the PS-II was localized in the thylakoid membrane proper, and extended to the surface of the loculus. This result is in harmony with the spatial localization of the PS-II complex particles on the EF face and the ES of the thylakoid membrane as demonstrated by the freeze fracture technique.

View full abstract

An unlabeled peroxidase-antiperoxidase (PAP) method on paraformaldehyde fixed and paraffin-embedded tissue sections was used to demonstrate the localization of retinol-binding protein (RBP) and prealbumin (PA) in the human kidney. The high specificity of the method was obtained by using highly purified primary and bridge antibodies after removing cross-reactive and heterophile antibodies which markedly contributed to background staining. The proximal convoluted tubular cells in the renal cortex were stained without exception with the antibodies to RBP and PA. The DAB reaction products, which appeared granular, were localized at the apical portions of the tubular cells. The finding coincides with the biochemical studies that RBP and PA were absorbed and catabolized in the proximal tubular cells after filtration through the glomeruli. From these results, it should be concluded that the specificity of the present method will provide a beneficial tool for the elucidation of RBP metabolism in the peripheral tissues.

View full abstract

The localization of Ca⁺⁺-activated adenosine triphosphatase (Ca⁺⁺-ATP-ase) activity was studied ultracytochemically in the transitional epithelium of the rabbit urinary bladder. The superficial cells contained two types of filaments, measuring 4-5nm and 6-10nm in diameter respectively. The former 4-5nm filaments are considered to correspond to actin filament, but the latter 6-10nm filaments seem to be composed of a little thinner, around 7nm, filaments and a little thicker, around 10nm, filaments. The thicker ones are located predominantly in the vicinity of the desmosomes and are inserted into them and are considered to be tonofilaments. The dense globular particles of the reaction products showing Ca⁺⁺-ATPase activity were observed in the filament-rich field. These products lay superimposed on the filaments or immediately lateral to them. Tonofilaments were, however, not positive for the activity.
These results suggest that the 4-5nm and probably 7nm filaments also, but not 10nm tonofilaments, are the contractile proteins. The deposits of the reaction products may correspond to the site of the cross-bridge of the contraction system. Functionally these filaments might serve for the change of the cell shape during the contraction-distension cycles of the urinary bladder.

View full abstract

Marked delay of fat absorption was observed in choline-deficient rats. The morphology of intestinal epithelial cells of choline-deficient rats was examined during fat absorption. By light microscopy, numerous lipid droplets remained in the apical zone of absorptive cells even 3hr after fat feeding. Ultracytochemically, the apical plasma membrane of these cells showed irregular microvilli with bleb formation and the Mg-ATPase and K-NPPase activities in the surface epithelium were decreased. Furthermore, absorptive cells in the choline-deficient state showed reduced Ca-ATPase (myosin ATPase) activity in the terminal web and other filamental bundles in close proximity to secretory vesicles throughout the cytoplasm. These findings were associated with poorly developed, disorganized microfilaments between the intracellular organelles and numerous large lipid droplets throughout the cytoplasm of absorptive cells. These lipid droplets often fused with each other and were large and irregular in shape near the Golgi complex. These observations indicate an altered state of contraction of microfilaments, accounting for impaired intracellular transport and discharge of chylomicrons by the intestine in choline-deficient rats.

View full abstract

The heptacosapeptide amide corresponding to the entire amino acid sequence of porcine gastrin releasing peptide (GRP), which had been isolated from porcine non-antral and intestinal tissue by McDonald et al. (5) and its amino acid sequence tentatively determined, was synthesized by Yajima et al. (11). Albino rabbits were immunized with this synthesized GRP and specific antiserum was obtained. Using an immunofluorescence antibody technique, cellular localization of GRP in the human stomach was examined.
GRP containing cells were clearly demonstrated in the mucosa of antrum of the stomach of a patient with a duodenal ulcer.

View full abstract

Cytochemical localization of glucose 6-phosphatase activity was studied in membranes of the endoplasmic reticulum and nuclear envelope of mouse hepatocytes. In periportal hepatocytes incubated in the reaction medium for 3min and centrilobular cells incubated for 15mim, the deposition of the reaction product for the activity was little, and therefore, relations between the reaction product and the membranes could be easily observed. In the rough endoplasmic reticulum, the reaction product was generally seen scatteringly on the inner surface of the membrane. In the smooth endoplasmic reticulum, the product appeared generally deposited in the cisternal space. In the nuclear envelope, the product was seen both on the inner surface of the outer nuclear membrane and in the perinuclear cisterna. Thus, the localization pattern of the reaction product was not common among the rough and smooth endoplasmic reticulum, and nuclear envelope. In periportal cells incubated for 5, 10 or 15min, the amount of the reaction product increased. However, the amount of the product was heterogenous in the lateral plane of the endoplasmic reticulum and nuclear envelope.
In the rough endoplasmic reticulum, the localization site of G6Pase activity is approximately the inner surface of the membrane.

View full abstract

Monoaminergic neuron systems have been studied in frontal, sagittal and horizontal sections of the bat brain by the indirect immunofluorescent method using antibodies to tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and serotonin (5-HT).
In general, the topographical distribution of monoaminergic neurons was found to be similar to that of the rat by Dahlstrom & Fuxe (3). However, some differences were found in the bat brain; namely the A3 group in the nucleus olivaris accessorius dorsalis and the A4 in the lateral part of the roof of the fourth ventricle were not observed.
The 5-HT-immunofluorescent varicose fibers and terminals were widely spread throughout the brain, especially in the supra-ependymal parts of the ventricular walls, several cranial nerve nuclei such as V, VII, and Xth nerve nuclei, and the catecholaminergic cell groups (A1, A2, A6, A9, A10). In addition, both the dopaminergic neurons and 5-HT neurons were observed in the nucleus raphe dorsalis and the ventral tegmental area caudal to the interpeduncular nucleus.

View full abstract

Living monkey peritoneal macrophages were incubated with cationized ferritin (CF) at 37°C. The endocytosis and the intracellular distribution of CF were investigated by transmission electron microscopy. CF was endocytosed via the coated and the uncoated pits within 5min. Phagocytosis-like process of the CF uptake was also observed. The endocytosed CF was found in round vesicles, tubular structures, large vacuoles, and the multivesicular bodies. Some of these vesicles were lysosomes. The CF containing vesicular structures tended to gather near and/or in the Golgi area. In the Golgi apparatus itself, however, only a small amount of CF was found in the stacked cisternae and the associated vesicles. The fate of the endocytosed CF is discussed.

View full abstract

Using both the biotinyl-lectins and avidin-ferritin (or HRP) conjugates, intracellular carbohydrates of guinea pig spermatids were stained on the ultrathin sections embedded in epoxy resin. Since the Golgi vacuoles, proacrosomal granules, acrosome cap, and acrosome collar of spermatids were densely stained by the “two-step method”, sugar residues in developing acrosomes of spermatids were systematically analyzed with seven different biotinyl-lectins, such as Con A, WGA, RCA, PNA, SBA, DBA and UEA. The reaction patterns of seven lectins to carbohydrates in acrosome were different from each other. The glycoproteins in both acrosome cap and acrosome collar of the step 5-7 spermatids were stained with RCA, Con A and PNA, while WGA, SBA and DBA reacted with carbohydrates in acrosome collar. These lectins did not react with carbohydrates in acrosome cap. Furthermore, the glycoproteins in proacrosomal vesicles and in the Golgi vacuoles surrounded by proacrosomal vesicles were heavily stained with RCA and Con A, but hardly stained with other Iectins. These results seem to suggest that the glycoproteins containing a large amount of D-mannose, D-glucose, and D-galactose in the sugar chains are synthesized throughout the whole stages of spermatids, while the glycoproteins containing N-acetylgalactosamine, terminal β-galactosamine, and N-acetylglucosamine in their sugar chains are mainly synthesized after the step 5 spermatids.

View full abstract

The interphase nuclei of petal cells of Tradescantia paludosa, which showed an active protein and ribonucleic acid (RNA) turnover but no detectable deoxyribonucleic acid (DNA) turnover, were assumed to be in metabolic interphase. These nuclei were estimated to have a dry mass of 0.25±0.03ng (mean and standard deviation) by X-ray microradiography, and 0.25±0.04ng by microinterferometry. The nuclear dry mass in this stage diverged wider (12.0-16.0%) than that in the interphase preceding meiosis (7.5%) and that in the zygotene stage of meiosis (3.4%). This divergency in dry mass is due mainly to an active turnover of nuclear acidic proteins, and may be an expression of divergent states in nuclear activity at the beginning of the differentiation period.

View full abstract

Two different techniques, enzyme-cytochemical and immunocytochemical, have been employed to localize alkaline phosphatase in the rat small intestine at light and electron microscopic levels.
In the absorptive cells of the duodenum and jejunum, alkaline phosphatase has been localized by the classical enzyme-cytochemical technique on the surfaces of apical microvilli and lateral membranes; on the other hand the direct immunoperoxidase method revealed some additional sites of the enzyme such as cisternae of rough endoplasmic reticulum and perinuclear spaces. Both enzyme- and immunocytochemistry failed to demonstrate the enzyme in the absorptive cells of the ileum. These immunocytochemical findings suggest that the absorptive cells in the proximal small intestine synthesize and transport the enzyme protein to their cell surfaces.

View full abstract

Differential staining of DNA replication in chromosomes was improved by adding urea treatment to the differential Giemsa method for sister chromatids. Using this improved method, the structure of chromosomes of Long-Evans rats was studied in correlation with G-, Q-, R- and C-bands as well as nucleolar organizer regions revealed by Ag-NOR and in situ molecular hybridization methods.

View full abstract

Both floating Vibratome sections from the following organs of rats, fixed in vivo by vascular perfusion; early and recovery stages of ethionine-induced fatty liver, secreting mammary gland, Gentamicin-injured renal cortex and resting adrenal cortex, and intact red cells of guinea pigs suspended in an isotonic buffer were treated, respectively, with the digestion medium containing each one of the following esterases; mold lipase, snake venom phospholipase and cholesterol esterase from microorganism. After digestion, the samples were immersed in 0.05-0.1% Pb(NO₃)₂, fixed in osmium buffer, dehydrated by Idelman's procedure, and finally embedded in epon. The derived fatty acids through enzymic digestions were detected in ultrathin sections as lead soaps, which were never seen in the duplicate control sections. Such a developed lead soap after digestion should indicate the location of substrate. This technique might be useful for the qualitative analysis of some mixedphased lipids in ultrastructure.

View full abstract

Fetal rat liver acid α-glucosidase was purified more than 20, 000 fold from the fetal rat liver 2 days before delivery. The disc gel electrophoresis of this preparation showed a single band of protein. The Michaelis constants of this enzyme were 3.3mM with maltose and 3.1mM with 4-methylumbelliferyl α-glucoside as substrates. Molecular weight measured by SDS electrophoresis and TOYOPEARL gel filtration was about 100, 000. The pH optima was 4.0 with 4-methylumbelliferyl α-glucoside as substrate and 3.7 with maltose as substrate.
Antibody was obtained by injection of this enzyme into rabbits with Freund's complete adjuvant. The anti-fetal acid α-glucosidase IgG showed the cross-reaction to the adult rat liver acid α-glucosidase. F(ab')₂ was separated by the filtration of Sephacryl S-200 column chromatography after digestion of anti acid α-glucosidase IgG pepsin and then F(ab')₂was treate d with 2-mercaptoethanol. Coupling of Feb', obtained by use of Sephacryl S-200 column chromatography after treatment of F(ab')₂ with 2-mercaptoethanol, to horseradish peroxidase was performed according to the method of Wilson and Nakane (14).
Light microscopical observation of the immunohistochemical localization of this enzyme in fetal rat and newborn rat liver showed small granular deposits of DAB reaction products in the cytoplasm.

View full abstract

A concurrent microchemical and topochemical study of DAB^* oxidation by isolated chloroplasts of Vicia faba under illumination was carried out. A new method for the quantification of the oxidation product of DAB was explored. The microchemical analysis by this method revealed that the photosystem responsible for the DAB oxidation was the PS-I. It also proved that endogenous active oxygen in the chloroplasts played only a little role in the oxidation. The topochemical analysis revealed that both the grana and the stroma membrane were positive in DAB photooxidation. The densitometric traces across the grana membranes in both surface and side views proved effective in locating the precise reaction sites. The partition layer showed a high degree of positive reaction. The thylakoid membrane proper also contained particles of positive reaction. The positive site extended to the thylakoid loculus to form protrusions. These results are in harmony with the localization of the PS-I complex particles on the PF face side of the thylakoid membrane as revealed by the freeze fracture technique.

View full abstract

The presence of acid phosphatase, β-glucuronidase and non-specific esterase activities were demonstrated in the clastic cells of Hassall's corpuscles in the guinea-pig thymus. These enzymes could conceivably play a role in dissolution of Hassall's corpuscles.

View full abstract

In the masseter and gastrocnemius three muscle fiber types can be distinguished histochemically by staining for succinate dehydrogenase activity. The relative contents of protein-bound SH groups in these three muscle fiber types were determined by quantitative cytophotometry by double staining for SH and protein. Significant differences in the relative SH contents of protein in different muscle fiber types were found in the gastrocnemius (type A<type B=type C), but not in the masseter. The relative SH content of protein in all muscle fiber types was higher in the masseter than in the gastrocnemius.

View full abstract

It is well known that mammalian spermatozoa utilize fructose as an energy source. In the mouse, the fructose is produced mainly in the seminal vesicle and in the coagulating gland. In this paper, the precise localization of sorbitol dehydrogenase in these organs was studied using an electron microscope. The results indicate that the enzyme is evenly distributed in the cytoplasmic matrix of epithelial cells of the seminal vesicles and the coagulating glands. The light microscopic observations which were performed by early investigators indicated that the distribution of sorbitol dehydrogenase in the epithelial cells of the seminal vesicle was very uniform, while that in the coagulating gland was restricted to the apical region of the epithelial cells. This apparent difference is related not to the local difference of the cytoplasm but to the amount of the cytoplasmic matrix. Since the morphoplasm. (nucleus, secretory granules, and the dilated cisternae) showed no reaction, the reaction in these regions might be relatively obscure under light microscopic observation.

View full abstract

Acetylcholinesterase (AChE) activity in the developing chick retina was histochemically studied by electron microscopy. The enzyme activity in the 1-day-old chick retina was confined within the ganglion, amacrine and horizontal cells as well as in the inner plexiform layer. In the inner plexiform layer reaction product was found in the intercellular spaces between nerve processes. Synapses in this layer did not always display reaction product. In the developing chick retina, AChE activity was found in the innermost layer of 6-day-old retina in ovo. The stage of the appearance of histochemically demonstrable AChE activity in the developing ganglion and amacrine cells mostly coincided with that of the beginning of morphological maturity of these cells. These findings indicate that the ganglion, amacrine and horizontal cells are functionally different from other types of neurons, and that synthesis of the enzyme starts at almost the same time as those cells begin to manifest the morphological characteristics of each cell type.

View full abstract

Uridine diphosphoglucose (UDPG) is conventionally used as a substrate for the histochemical demonstration of glycogen synthetase activity in mammalian tissues. In the present study, glycosyl nucleotides other than UDPG, guanosine diphosphoglucose (GDPG) and thymidine diphosphoglucose (TDPG), acted as the substrate to demonstrate the glycogen synthetase activity in rat skeletal muscle fibers. The histochemical reaction using GDPG and TDPG was very similar in its location but not in its intensity to the UDPG one. Furthermore, the different size and shape of the new glycogen particles synthesized from GDPG and TDPG by the enzyme activity were observed according to the type of skeletal muscle fibers under the electron microscope. The results presented here indicate that GDPG and TDPG as well as UDPG act as glucosyl donors for new glycogen synthesis through the glycogen synthetase activities in rat tissues, and characteristic glycogen particles can be observed under light and electron microscopic conditions.

View full abstract

The metalloproteins in the liver of p-dimethylaminoazobenzene (DAB)-treated rats were analyzed by means of immunohistochemical and electron spin resonance (ESR) techniques in comparison with normal or carbon tetrachloride-damaged livers. Immunohistological method revealed intensive distribution of serum ceruloplasmin and transferrin in DAB induced tumor tissues. Concomitantly, a remarkable increase was also noted in the ESR signals of both cupric ion and non-heme ferric ion of rhombic high-spin form, which were ascribed to these metalloproteins. Their biological implications in malignancy are discussed.

View full abstract

This study was carried out to elucidate the mechanism of an autophagy of peroxisomes seen in hepatic parenchymal cells in the CPIB-treated rat liver followed by partial hepatectomy or by glucagon administration. The catalase activity demonstrated cytochemically by the diaminobenzidine reaction was used as a marker for peroxisomes and the acid phosphatase activity demonstrated by the lead method for lysosomes.
The large size and number of peroxisomes induced by the CPIB administration in the rat hepatic parenchymal cell showed a gradual decrease in number after partial hepatectomy with concomitant increase in the number of lysosomes of various sizes including autophagolysosomes. Autophagoly-sosome formation involving peroxisomes was also evident in the hepatic parenchymal cells in the CPIB-treated and glucagon-administered rat liver. Furthermore, participation of the lysosomal wrapping mechanism in autophagocytosis of peroxisomes was frequently observed.
It was concluded in the present investigation that the lysosome participates in the catabolism of peroxisomes observed in the CPIB-treated rat liver followed by partial hepatectomy or glucagon administration.

View full abstract

In order to clarify the distribution of cholesterol in plasma- and cytomembranes of secretory cells of the anterior pituitary and adrenal medulla, freeze-fracture images of the filipin-treated tissues were observed.
The distribution of cholesterol in all the secretory cells of these organs shows almost the same pattern. The filipin-sterol complexes are distributed somewhat heterogeneously but in moderately high density on the fractured plasma membranes of all the cells examined. The small shallows on the plasma membrane, corresponding to coated pits and showing aggregates of membrane particles (MP), lack the complexes. The limiting membranes of almost all the secretory granules are very rich in complexes.
The membranes of rough endoplasmic reticulum, nucleus, mitochondria, and Golgi apparatus, which are considered to contain rich enzymatic protein and have active functional properties, are very poor in distribution of cholesterol, though small clusters consisting of a few complexes are scattered in these organelles except for mitochondria. In the traps-side of the Golgi apparatus, where the immature secretory granules arise, the complexes are relatively denser in distribution as compared with those of the cis-side.

View full abstract

Immunohistochemical studies have demonstrated that in the adult rat and human anterior pituitary glands that one cell might be devoted to the production of one hormone with the exception of FSH and LH. In addition, the intermediate lobe cells are exclusively positive for ACTH. In studies of rat and human fetal pituitary glands, ACTH was the first hormone to appear in both anterior and intermediate cells, followed by other hormone-producing cells. As the hypothalamic tropic hormones appear later than the corresponding pituitary hormones, it was considered that the pituitary cells might differentiate toward hormone production independently of the hypothalamus and that hypothalamic tropic factors might be necessary to maintain or promote hormone production. This is supported by studies on human anencephalic pituitaries and cultured rat pituitary primordial cells.

View full abstract