Showed Reaction Product Research Articles

Calcium-activated adenosine triphosphatase and gap junctions in rat epidermis.

Ca++-activated adenosine triphosphatase (Ca++-ATPase) in the epidermal cells of rat skin was demonstrated with the method of Ando et al. (1981), and the presence of enzyme activity on the epidermal gap junctions was obtained. Furthermore, the gap junctions showing enzyme activity were examined morphometrically under a digigramer model-G (Muto Kogyo Co. Ltd., Japan).The electron microscopic observation of epidermis showed reaction products of Ca++-ATPase on the entire cell surface continuously except desmosomes and basal cell membranes facing the basal laminae in the stratum Malpighii and a few reaction products in the stratum granulosum. With keratinocytes differentiating, activities seemed to be diminishing. The reaction products were also found on the cytoplasmic side of the gap junctional membranes. Gap junctions showing the reactions were more often detected in the upper portion of the stratum Malpighii and/or in the lower portion of stratum granulosum of the rat pad epidermis.1. The findings of Ca++-ATPase in the rat epidermis might reflect changes in the intracellular level of calcium which occur during the proliferation and the differentiation of epidermal cells.2. The unequal distribution of the epidermal gap junctions appears to be involved in the synchronous differentiation process and in the maintenance of the epidermal thickness.

A phosphatase activity and a synaptic vesicle antigen in multivesicular bodies of frog retinal photoreceptor terminals.

Previous work has suggested that multivesicular bodies participate in endocytosis and membrane cycling at nerve terminals, including the presynaptic terminals of retinal photoreceptors. We now have found that multivesicular bodies located in the presynaptic terminals of photoreceptors in retinae of Rana pipiens show reaction product in preparations incubated to demonstrate phosphatase activity at pH 5, using cytidine monophosphate as the substrate. Evidently, multivesicular bodies in photoreceptors can possess at least some hydrolytic enzymes during their sojourn in the terminals. We have also found that the multivesicular bodies in frog retinal photoreceptor terminal stain, immunocytochemically, for the presence of SV2, an antigen of synaptic vesicles. This observation supports the suggestion that, along with the extensive, repeated reuse of membrane components for synaptic vesicle recycling, there is some incorporation of the components into structures that are potentially degradative.

Ultrastructural localization of 2-naphthylthiol acetate nonspecific esterase in human blood cells and leukemic cells

Nonspecific esterase activity was localized ultrastructurally within normal and leukemic hematopoietic cells by the use of 2-naphthylthiol acetate (NTA) as a substrate. NTA esterase activity was identified in all cell lines, although mononuclear phagocytes contained the most abundant activity. Monocytes, macrophages, young granulocytes, eosinophils, basophils, megakaryocytes, and platelets showed reaction products primarily associated with the membranes of cell granules, mitochondria, rough endoplasmic reticulum, and perinuclear cisternae. Lymphocytes demonstrated focal uneven staining in perinuclear cisternae and endoplasmic reticulum, with only occasional cytoplasmic reactions. Erythroblasts showed the most distinctive staining pattern with predominance of large amounts of reaction product within the perinuclear space in a ring-like distribution. Examination of the staining pattern in 42 cases of leukemia and 2 cases of malignant lymphoma demonstrated only limited usefulness as a diagnostic aid.

Cytochemical localization of Ca2+-Mg2+ adenosine triphosphatase in rat incisor ameloblasts during enamel secretion and maturation.

A modified Wachstein-Meisel medium containing lead or cerium as capturing ions was used to localize Ca2+-Mg2+ adenosine triphosphatase (ATPase; EC 3.6.1.3) in rat incisor ameloblasts during enamel formation. Sections representing different developmental stages were processed for electron microscopic cytochemistry. Distribution and intensity of the observed reaction product, which was almost exclusively associated with cell membranes, varied according to the stage of enamel formation. During the secretory stage, intense reaction product was evident along the entire plasma membrane of ameloblasts and papillary cells. The early transitional ameloblasts showed reaction product on their proximal and lateral cell membranes, but not distally. In late transitional (pre-absorptive) ameloblasts, distal cell membranes exhibited intense reaction product. During enamel maturation, smooth-ended ameloblasts showed reaction product proximally and laterally, but not distally. Ruffle-ended maturative ameloblasts exhibited intense reaction product along their lateral and distal membranes. The intensity of the latter was decreased but not eliminated by levamisole. In the transition from smooth-ended to ruffle-ended cells, the reaction product became evident distally, concomitant with the appearance of cell membrane invaginations. These data are consistent with a possible role for Ca2+-Mg2+ ATPase in controlling calcium availability at the enamel mineralization front.

Enzyme histochemistry of ventricular choroid plexus. Localization of Ca++-ATPase activities.

Ultracytochemical localization of Ca++-adenosinetriphosphatase (ATPase) activity was investigated in the choroid plexus of the rat fourth ventricle. A newly devised lead citrate method (Ando et al., 1981) was employed for the detection of this enzyme. Electron dense reaction products for Ca++-ATPase activity were shown on the external surfaces of apical plasma membranes, including those of the microvilli and cilia, and on the external surfaces of basal plasma membranes, especially those of the basal infoldings. The gap junctions also showed reaction products on the cytoplasmic side of the plasma membranes. Among the intracellular organelles, activity was seen exclusively on the cristae of mitochondria. From these findings, it can be suggested that Ca++-ATPase participates in Ca++ transport and in the regulation of cerebrospinal fluid secretion in association with intracellular Ca++ and Na+, K+-ATPase.

Occurrence and ultrastructural localization of duck hepatitis B virus in the liver of ducks after experimental infection.

A sequential study was performed to investigate the occurrence and localization of duck hepatitis B virus in the liver of domestic ducks utilizing the indirect immunoperoxidase method and electron microscopy. Seventeen ducklings were injected intravenously with duck hepatitis B virus-positive serum within 24 hr after hatching and were subsequently sacrificed on the 2nd, 3rd, 4th, 5th, 27th and 44th day after injection. Nine ducklings were not injected and were used as a negative control. Duck hepatitis B virus DNA by spot hybridization using a [3P]-labeled probe occurred in trace amounts on the 2nd day and in large amounts on the 4th day after inoculation. Immunoreactivity for DHBV was seen in the hepatocytes, sporadically on the 2nd day and diffusely on the 4th day, and also in the biliary epithelial cells on the 27th day. Both kinds of cells revealed staining in the cytoplasm but not in the nucleus. Virus particles were recognized by electron microscopy in the hepatocytes beginning on the 4th day. The hepatocytes had many incomplete virus particles, 40 to 61 nm in diameter, and a few complete virus particles, 40 nm in diameter, in the cisternae of the rough and smooth endoplasmic reticula. Such particles and the endoplasmic reticulum showed reaction products for duck hepatitis B virus by immunoelectron microscopy. There were clusters of core particles, 27 nm in diameter, in the hyaloplasm around peroxisomes where an assembly of cores appeared to occur. No conspicuous virus particles were recognized in the biliary epithelial cells. The similarities and differences in virus localization between duck hepatitis B virus and hepatitis B virus are discussed.

Immunocytochemical localization of a rhodopsin-like protein in the lipochondria in photosensitive neurons of Aplysia californica.

Polyclonal antibodies directed against squid opsin were used in immunocytochemical and immunoblot experiments to identify a rhodopsin-like protein in photosensitive neurons of Aplysia. Aldehyde-fixed abdominal and cerebral ganglia were embedded in paraffin for peroxidase anti-peroxidase analysis or used whole for immunofluorescence studies. Ganglia were embedded in Lowicryl K4M for electron-microscope immunocytochemistry. In both the cerebral and abdominal ganglia, light-microscope immunocytochemical results showed reaction product deposited around the neuronal cell periphery corresponding in position to the lipochondria. In the abdominal ganglion, the giant cell R2, located in the right rostral quarter, and neurons in the right caudal quarter were consistently labeled with anti-opsin. Electron-microscopic studies demonstrated ferritin-labeling of the lipochondria in R2 and other immunoreactive neurons. Immunoblot analysis of R2 and cerebral neuron extracts was used to identify two prominent immunoreactive protein bands at 85 000 and 67 500 molecular weight.

Light and electron microscope localization of beta-glucuronidase activity in the stomach and digestive gland of the marine gastropod Littorina littorea.

An azo dye technique was used to investigate localization of the acid hydrolase, beta-glucuronidase, at light and electron microscope level in the stomach and digestive gland of the marine periwinkle Littorina littorea. Activity for beta-glucuronidase was located principally within digestive cells of the digestive gland and also associated with the microvillous border and epithelial cells lining the stomach. At the light microscope level all digestive tubules showed activity which appeared essentially restricted to the large heterolysosomes of the digestive cells. However not all digestive cells showed activity. In the electron microscope, reaction product was apparent in all types of macrovesicle in the digestive cells although not all stained positively. Heterophagosomes typically showed reaction product around their periphery or associated with the electron opaque contents. Activity was commonly seen around the apical edge of heterolysosomes where merging of heterophagosomes into heterolysosomes was apparent. Reaction product was commonly located within small electron lucent vesicles which lined the internal membrane of the heterolysosomes but sometimes also associated with flocculent, electron opaque contents. In the stomach dense clusters of reaction product were visible in lysosomes in the basal region of the epithelial cells and in the large granular inclusions of the secretory cells.

Prefertilization ovule development in Capsella: the dyad, tetrad, developing megaspore, and two-nucleate gametophyte

Ovules of Capsella bursa-pastoris at the dyad and tetrad stages of meiosis and at the megaspore and two-nucleate stages of the gametophyte were studied with the electron microscope. The cells of the dyad and tetrad are separated by aniline blue fluorescent cross walls and receive all types of organelles and autophagic vacuoles that were present in the meiocyte. Autophagic vacuoles enclose ribosomes and organelles and show reaction product for acid phosphatase. Autophagic vacuoles and some plastids are absorbed into the enlarging vacuoles of the growing megaspore. Other plastids appear to survive meiosis and there is no evidence for their de novo origin. Some mitochondria appear to degenerate in the enlarging megaspore but others look healthy and there is no evidence for the de novo origin of mitochondria. The nucleolus of the developing megaspore becomes very large and the cytoplasm is extremely dense with ribosomes. The cell wall is thickened by an electron-translucent, periodic acid – Schiff negative, aniline blue fluorescent material and contains plasmodesmata that link the megaspore with the nucellus. The plasmalemma of the growing megaspore produces microvilluslike extensions into this wall that disappear with the formation of the two-nucleate gametophyte. Plasmodesmata disappear from the cell wall at the four-nucleate stage.

Peroxisomes in the nervous system of Aplysia californica: a cytochemical study.

We have studied the distribution of peroxisomes in the abdominal ganglion of Aplysia californica using electron microscopic cytochemical methods. Reaction product for catalase was observed in small ovoid or dumb-bell-shaped bodies in the perikarya of many of the neurons. The abundance of these catalase-reactive peroxisomes is considerably greater than is the case in vertebrate neurons. While the non-neuronal cells of the Aplysia abdominal ganglion do contain appreciable peroxisome populations, there were few peroxisomes in glial cytoplasm directly adjacent to the perikarya, again contrasting with vertebrate ganglia in which the satellite cells are a principal site of peroxisomes. Peroxisomes are present throughout the perikaryal cytoplasm. In the regions in which lipochrome granules abound, peroxisomes are frequently seen closely associated with these granules; glycogen is abundant nearby. The association of peroxisomes, lipochrome granules and glycogen is interesting in view of the propinquities of peroxisomes to lipid droplets and lipofuscin granules reported for non-neuronal vertebrate tissues, and in view of the growing evidence indicating that some of the roles of peroxisomes are in lipid metabolism and in gluconeogenesis. Some of the lipochrome granules themselves show reaction product in ganglia incubated to demonstrate catalase activity and some react in tissue incubated to demonstrate acid phosphatase activity. Such observations suggest that the enzymatic capacities of the lipochrome granules merit further studies, and that the granules may be of complex or heterogeneous nature.

Immunoelectron microscopic demonstration of prostatic acid phosphatase in human hyperplastic prostate

Immunoelectron microscopic studies were done on prostatic tissues obtained from patients with benign hyperplasia. Rabbit IgG-peroxidase conjugate against purified human prostatic acid phosphatase band 2 (HPAP-2) was used for studies. Under the light microscope, the columnar secretory epithelia of prostatic glands showed different intensity and distribution of immunostaining whereas the basal cells were unstained. Under the electron microscope, the secretory epithelial cells often showed electron-dense reaction product in the Golgi apparatus and secretory vesicles and vacuoles, and only sparingly in the cisternae of nuclear envelope and rough ER. Sometimes, fusion of secretory vacuolar membrane and plasma membrane and discharge of the vacuolar contents into the extracellular space were noted. The surfaces of microvilli at the apical portion of the columnar epithelia and the lumen of the glandular acini always showed reaction product. These findings suggest that HPAP-2 may be synthesized in the rough ER and transported to the Golgi apparatus where it is concentrated and transferred to the secretory vesicles and vacuoles. HPAP-2 is finally discharged into the extracellular spaces through exocytosis, a secretory mechanism similar to that of other secretory proteins.

Immunocytochemical study on photoreceptor cell differentiation in the cultured retina of the chick

Photoreceptor cell differentiation was investigated in a dissociated monolayer culture of chick embryonic retinas with electron microscopic immunohistochemistry. The antibody was raised against bovine rhodopsin purified on SDS-polyacrylamide gel electrophoresis. In the developing retina, immunoreactivity was first recognized on the 14th day of incubation and was localized on the plasma membrane of the growing inner segment. On the 16th day, immunoreactivity was observed on some differentiating outer segments but not on inner segments. In the culture from 6 1 2 - day-old embryonic retinas, immunoreactivity was found on the 7th day of culturing on the plasma membrane of large-sized neurons. Electron microscopic observations confirmed that such stained cells showed reaction product on the plasma membrane, and that they displayed fine structures characteristic of intact photoreceptor cells. They had a number of microvillous processes and often one thick process, both of which were intensely stained. Outer segment formation, however, was not observed in the present monolayer culture. These results indicate that opsin synthesis and its transport to the plasma membrane begins prior to and probably independently of outer segment formation.

Effect of concanavalin A on lymph node macrophages: stimulation of endocytic cisternae

Incubation of isolated lymph node macrophages with concanavalin A (Con A) resulted in a dense and continuous labeling of the plasmalemma and filopodia which were closely adherent to each other and the cell surface. Within a short time period (3-5 min), membranes of the closely apposed filopodia became invaginated into the cytoplasm to form numerous interconnecting cisternae. After 10 min the system of internalized membranes had migrated into the deeper cytoplasm and was closely associated with numerous actin filaments and other components of the cytoskeleton. The internalized plasmalemma remained in the cytoplasm up to 24 hr without fusing with lysosomes. Concomitant with plasmalemmal invagination and the formation of cisternae there were also changes in the Golgi apparatus. These appeared in the form of hypertrophied Golgi saccules and the accumulation of numerous vesicles around the Golgi. Treatment of isolated lymph node macrophages with either succinylated Con A, alpha-methyl-D-mannoside, or ferritin particles alone failed to produce the cisternal structures. The results suggested that the tetravalency of Con A may be responsible for the binding of adjacent Con A-labeled membranes to each other and for maintaining a crosslinking of membranes during invagination and internalization. It is suggested that this process of extensive membrane internalization represents a specialized form of endocytosis. At 24 hr after incubation with Con A, cisternal structures in close proximity to the Golgi vesicles showed signs of degradation. By 48 hr there was a breakdown of cisternal membranes with a release of Con A marker particles into large phagocytic vesicles, which also showed reaction product for acid phosphatase, suggesting a fusion with lysosomes.

Electron microscopic histochemistry of alkaline phosphatase activity in the developing chick retina

The present paper describes ultrastructural localization of non-specific alkaline phosphatase (ALPase) activity in the chick retina during late embryonic development and after hatching. In the newly-hatched chick retina, intense ALPase activity was found in the outer plexiform layer (OPL), and a less intense reaction was found in the ganglion cell layer and at the outer limiting membrane. Electron microscopic histochemistry revealed that bipolar and horizontal dendrites within the OPL displayed intense reaction product along their plasma membranes. Only horizontal cells showed reaction product on almost their whole circumference. Müller cells also showed reaction product on the plasma membrane in the ganglion cell layer and on the microvilli at their apical portion. The reaction product in the OPL was first recognizable in the retina of the 13- to 14-day-old embryonic chick, and its intensity increased during further development. Little reaction product was found in Müller cells at the embryonic stage. The function of ALPase activity is discussed in relation to the development of the OPL and the nutrient supply for the neural retina.

Immunohistological analysis of surfactant-apoprotein in the bronchiolo-alveolar carcinoma.

Fifty-five cases of bronchiolo-alveolar carcinoma were examined immunohistochemically using mono-specific antisurfactant apoprotein IgG obtained from a rabbit immunized with monkey surfactant preparations. Formalin-fixed and paraffin-embedded lung tissues were stained by an immunoperoxidase method. Antibody stained the normal and hyperplastic alveolar type II pneumocytes, but did not stain bronchial epithelium or other lung cells. In tumor tissue, 26 (47.3%) of 55 cases were positively stained in the cytoplasm, and 15 showed reaction products in both the cytoplasm and intranuclear regions. By electron microscopy osmiophilic lamellar bodies and microvilli on the free surface, thought to be characteristic of type II pneumocytes, were seen in tumor cells (2 cases). In the five cases, the nuclei contained branching tubular inclusions. The results of this study support the idea that certain bronchiolo-alveolar carcinomas originate from type II pneumocytes, the intranuclear inclusions may represent an abnormal proliferation of nuclear membranes containing surfactant-apoprotein.

Ultrastructural localization of phosphatases in the testes of the domestic fowl: acid phosphatase and thiamine pyrophosphatase.

ACPase and TPPase activity has been examined in the germinal epithelium of the testes in the domestic fowl. ACPase activity in spermatogonia and spermatocytes was confined to the Golgi complex. In spermatids ACPase activity was seen in the endoplasmic reticulum and nuclear envelope in the phase I and especially in the phase II (the elongating phase). This activity gradually decreased during the next phase III (the elongating phase). This activity gradually decreased during the next phase III, and had disappeared in the final phase IV. The membrane body showed ACPase reaction in the small peripheral vacuoles and cisternal structures surrounding large central vacuoles. ACPase was also present in vesicles surrounding the developing tail. Late spermatids showed an abundance of autophagic vacuoles which had a complex array of ACPase positive delimiting membranes. In Sertoli cells ACPase activity was predominant in the lysosomes. TPPase activity was seen in the cisternae of the Golgi complex in spermatogonia and spermatocytes. In spermatids activity was present in the endoplasmic reticulum during the phase II, but it is lost in later stages. The smaller vacuoles and cisternal structures in the membrane body also showed reaction products. According to the present results it is thought likely that the smaller vacuoles and cisternal structures of the membrane body are of endoplasmic reticulum origin. The autophagic vacuoles in spermatids and the lysosomes of Sertoli cells are considered responsible for the degradation of residual bodies cast off by spermatids.

Ultrastructural localization of coenzyme A phosphatase (CoA-Pase) activity to the GERL system in secretory ameloblasts of the rat incisor.

Enzymatic hydrolysis of the monoester phosphate group from coenzyme A (CoA) was studied in rat incisor ameloblasts by incubating specimens from glutaraldehyde-fixed teeth in a cytochemical medium prepared with acetyl-CoA as substrate and lead ions as capture agent for phosphate. Ameloblasts incubated for 1 hr at 37 degrees C and at pH 5.0 in this medium showed reaction product localized almost exclusively along the trans (mature) aspect of the Golgi apparatus within a network of small granules and interconnecting tubular channels that comprise the GERL system in this cell. Reaction product was otherwise seen in trace amounts only within some Golgi saccules, a few lysosomal dense bodies and, in rare instances, within an occasional focal area of the endoplasmic reticulum. No selective staining of the GERL system was seen in control ameloblasts incubated at either pH 7.2 or pH 9.0 with acetyl-CoA as substrate, or incubated at pH 5.0 with dephospho-CoA as substrate. Control experiments at pH 5.0 also revealed that reaction product selectively stained the GERL system in ameloblasts when other molecules resembling CoA were used as substrate (e.g., crotonyl-CoA, 3'-NADP+), but not when adenosine 3'-monophosphate (3'-AMP) was used as substrate. That is, ameloblasts incubated at pH 5.0 with 3'-AMP showed heavy deposits of reaction product at many sites throughout the cell, including most lysosomal dense bodies, the Golgi saccules, the GERL system, most secretory granules, the nucleus, and extensively throughout the endoplasmic reticulum. These findings suggest that the GERL system of ameloblasts contains a CoA-specific phosphatase activity that may function to convert CoA to dephospho-CoA at acid pH. Biochemical studies included with this investigation further indicate that CoA-Pase activity saturates at exceptionally low concentrations of substrate (KM = 30 microM CoA) compared to other acid-dependent phosphatases.

Central projections of muscle afferents from the sternomastoid nerve in the rat

Perikarya and central endings of muscle afferents of the sternomastoid nerve of the rat were investigated using the horseradish peroxidase (HRP) method in its modification by Mesulam. Application of an aqueous solution of HRP to the cut sternomastoid nerve was followed by heavy labeling of cell bodies in ipsilateral spinal ganglia C3 and C4. In addition, a number of peripheral and central processes of spinal ganglion cells clearly showed reaction product. Labeled structures in the spinal cord and medulla oblongata were regarded as axons and/or terminals of sternomastoid nerve primary afferents because the motor root to this nerve had been interrupted before the application of HRP. Analysis of serial sections and mapping of all labeled nervous structures in the CNS revealed many stained axons and terminals in the medial parts of dorsal and ventral horns as well as in the medial part of zona intermedia in the segments C1–C3. Clearly labeled zones were also seen in the central and lateral areas of the ventral horn. These zones correspond to the location of the motor nuclei for the sternomastoid and infrahyal muscles, partly also for the longus colli and splenius capitis muscles. The labeled area in the zona intermedia includes the field of the nucleus cervicalis centralis, which was shown by other authors to receive input from primary afferents of the neck region as well as from vestibular nuclei via fasciculus longitudinalis medialis, and which in turn sends many fibers into the cerebellum. Label was also found in many axons of the fasciculus cuneatus and in terminals spreading over the whole nucleus cuneatus lateralis. Here, close relations of those terminals to perikarya were seen. Only weak labeling was found in laminae I–III of the upper three spinal cord segments.

Fate of horseradish peroxidase in the secretion zone of the rat incisor enamel organ

Adult CDF albino rats were killed from 10 min to 6 hr after a single intravenous dose of HRP. Experimental and control tissues were reacted for peroxidase activity and processed for light and electron microscopy. At 10 min, all extracellular spaces of the secretion zone showed reaction product. A reaction was also seen around Tomes' processes and in a layer of enamel spaces in the region of thin enamel. At later time intervals, reactions around Tomes' processes were also seen in regions of thicker enamel. Tracer was located preferentially at the growth fronts of rod and interrod enamel, and also diffused for some distance into enamel. From 2 to 6 hr, the enamel over the transition zone became heavily labeled. The tracer penetrated for more than 90 μm into the enamel and was localized mainly in the interrod enamel. Droplets of dense stippled material in the extracellular spaces between Tomes' processes did not mix with tracer, but sites which contain a light stippled material in the controls (extracellular spaces, vesicles within ameloblasts) showed a reaction. It is concluded that (1) the basal terminal bars of secretory ameloblasts do not impede the flow of large molecules, (2) the apical terminal bars are permeable in early secretion, become increasingly tight as secretion progresses, and are again permeable in the transition zone, (3) ameloblasts can shuttle large extracellular molecules towards the enamel growth fronts, (4) large molecules can diffuse into enamel; rod and interrod enamel differ with regard to the diffusion of large molecules, (5) ameloblasts phagocytose significant amounts of light stippled material. The possibility is considered that extracellular enamel precursor molecules move preferentially towards the enamel growth fronts, perhaps by a mechanism involving membrane flow, and diffuse through enamel in similar fashion as HRP.

Ultrastructure of acetylcholinesterase containing striato-nigral neurons transporting HRP retrogradely

The fine structure of striato-nigral efferent neurons, retrogradely labeled with HRP, was studied. The neuron showed a medium sized ovoid or triangular perikaryon. Short or medium lengthened granular endoplasmic reticula, having a tendency to wind and pile up in stacks were observed. This neuron corresponds to a type III neuron according to our classification [4,5]. The neuron showed reaction products, indicating AChE activity, in the granular endoplasmic reticula and plasma membrane.