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Photodynamic therapy (PDT) using the photosensitizer talaporfin sodium (talaporfin) is a new mode of treatment for cancer. However, the metabolic mechanism of talaporfin has not been clarified. Thus, we investigated the uptake, transportation, and elimination mechanisms of talaporfin in carcinoma and sarcoma. The results showed that talaporfin co‐localized in early endosomes and lysosomes. Talaporfin uptake was via clathrin‐ and caveolae‐dependent endocytosis and a high amount of intracellular ATP was essential. Inhibition of lysosomal enzymes maintained intracellular talaporfin levels. Inhibition of K‐Ras signaling reduced talaporfin uptake in carcinoma and sarcoma cell lines. Talaporfin was taken up by clathrin‐ and caveolae‐dependent endocytosis, translocated from early endosomes to lysosomes, and finally degraded by lysosomes. We also demonstrated that ATP is essential for the uptake of talaporfin and that activation of K‐Ras is involved as a regulatory mechanism. These results provide new insights into the metabolism of talaporfin in cancer cells for the enhancement of PDT for carcinoma and sarcoma., Intracellular talaporfin was co‐localized with early endosomes and lysosomes but not with mitochondria in osteosarcoma cells.

Photodynamic diagnosis/therapy (PDD/PDT) are novel modalities for the diagnosis and treatment of cancer. The photosensitizer protoporphyrin IX is metabolized from 5-aminolevulinic acid (5-ALA) intracellularly, and PDD/PDT using 5-ALA have been approved in dermatologic malignancies and gliomas. However, the molecular mechanism that defines the efficacy of PDD/PDT is unknown. In this study, we analyzed the functions of ATP-binding cassette (ABC) transporters in PDD using 5-ALA. Most of the human gastrointestinal cancer line cells examined showed a homogenous staining pattern with 5-ALA, except for the pancreatic cancer line PANC-1, which showed heterogeneous staining. To analyze this heterogeneous staining pattern, single cell clones were established from PANC-1 cells and the expression of ABC transporters was assessed. Among the ABC transporter genes examined, ABCG2 showed an inverse correlation with the rate of 5-ALA-positive staining. PANC-1 clone #2 cells showed the highest level of ABCG2 expression and the lowest level of 5-ALA staining, with only a 0.6% positive rate. Knockdown of the ABCG2 gene by small interfering RNAs increased the positive rate of 5-ALA staining in PANC-1 wild-type and clone cells. Interestingly, PANC-1 clone #2 cells showed the high sphere-forming ability and tumor-formation ability, indicating that the cells contained high numbers of cancer stem cells (CSCs). Knockdown or inhibition of ABCG2 increased the rate of 5-ALA staining, but did not decrease sphere-forming ability. These results indicate that gastrointestinal cancer cell lines expressing high levels of ABCG2 are enriched with CSCs and show low rates of 5-ALA staining, but 5-ALA staining rates can be improved by inhibition of ABCG2.

Atomic force microscopy (AFM) has been systematically used to image the surface of a femoral head made of alumina/zirconia composite (henceforth referred to as zirconia-toughened alumina, ZTA), in comparison with two types of commercially available femoral heads made of monolithic zirconia. AFM experiments were conducted before and after in vitro exposure in water moist environment. All materials contained zirconia partly stabilized with yttria. AFM observations were performed on wide areas of several heads of each type (i.e., in the order of several tens of μm2), in order to ensure statistical reliability in the topologic measurements. Tetragonal-to-monoclinic phase transformation, which was quantitatively characterized by confocal Raman spectroscopy, showed significant difference among the investigated samples. Such differences were similarly found with respect to both roughness and time needed for topologic changes to occur. Variation of topologic statistic parameters, such as skewness and kurtosis of surface height histograms, confirmed the main impact of zirconia grain size on the environmental stability of the head surface, the finer the grain size the higher the stability.

The permeability of higher molecular weight substances was investigated in mouse chorioallantoic labyrinthine hemotrichorial placenta, using horseradish peroxidase as a tracer. At the same time, ultrastructural localizations of some important enzymes, such as alkaline phosphatase (ALP), acid phosphatase (ACP), Ca++-ATPase and guanylate cyclase were elucidated in this organ by means of the enzyme-cytochemical technique. Peroxidase easily entered the space between layers I and II, and no penetration of this tracer beyond layer II was observed. The reaction products for ALP activity were found mainly on the maternal side of the plasma membrane of the layer II tro-phoblast. ACP activity was confined to the lysosomes of this layer II cell. In short, peroxidase stopped at the cell surface of the layer II trophoblast, and both ALP and ACP coexisted in this layer II cell. These observations strongly suggest that the layer II trophoblast, especially the surface plasma membrane of this cell, may have an important role in regulating the materno-fetal transfer of substances in mouse chorioallantoic placenta.

The effects of motion on the anterior cruciate ligament were examined using knee joints. When the knees of rats and rabbits subjected to motion in a horseradish peroxidase (HRP) solution were compared with those undisturbed in an identical solution, it was found that a greater quantity of HRP had infiltrated into the exercised knees. We concluded that motion facilitates nutrient infiltration into the anterior cruciate ligament.

Human scalp hair is important as a diagnostic clue to many diseases, in medical jurisprudential investigations, and also as a subject of cosmetic treatments. While many ultrastructural studies of the human hair root including the hair follicle have been reported, few studies have been done on the human hair shaft. We report here the ultrastructure of human scalp hair shafts prepared by a rapid-freezing technique followed by freeze-substitution fixation that allows the observation of fine cell structures. Healthy scalp hair shafts from Japanese females 12–13 years of age were rapid-frozen and then freeze-substituted in OsO4-acetone. In addition, this technique was applied to the study of some changes of the hair shafts (i.e., hair damaged by thioglycolic acid cold permanent waving and white hair). By this method, the hair shaft was rapid-frozen throughout without appreciable ice damage although the hair shaft was nearly 100 μm in diameter. The rapid-freezing technique resulted in excellent preservation of the ultrastructure of the hair shafts: lamellar structures in the cuticle and fine fibrous ultrastructures in the cortex were observed without chemical treatments. Thioglycolic acid treatment affected the ultrastructure of both the cuticle and the cortex. Except for the absence of melanin granules, no significant differences in the ultrastructure were observed between white hair and black hair. The rapid-freezing technique followed by freeze-substitution fixation appears to be the most reliable approach for the morphological evaluation of fully keratinized cells and tissues. Anat. Rec. 251:406–413, 1998. © 1998 Wiley-Liss, Inc.

Glucose-6-phosphatase is generally accepted as a functional component of rough endoplasmic reticulum and has been histochemically examined in many organs. The aim of this study is to know the ultracytochemical localization of glucose-6-phosphatase in each type of hormone-producing cell constituting the anterior pituitary gland in the rat. Pituitaries of male Sprague-Dawley rats were perfused with 1.5% glutaraldehyde from the left ventricles. After buffer washing 40 microns sections were incubated in the medium of Hugon et al. for 60 min at 37 degrees C. The sections were then postfixed with 1% osmium tetroxide, embedded in epoxy resin and observed under an electron microscope. The reaction product for glucose-6-phosphatase was observed in the lumen of rough endoplasmic reticulum and nuclear envelope of all anterior pituitary cells. The enzyme activities in thyroid-stimulating hormone-producing cells and luteinizing hormone/follicle-stimulating hormone-producing cells (LH/FSH cells) were stronger than those in growth hormone-producing cells and prolactin-producing cells; adrenocorticotropic hormone-producing cells and folliculo-stellate cells presented intermediate activity. In LH/FSH cells, the activity in dilated cisternae of endoplasmic reticulum had weaker density than that in flattened cisternae. In addition, substantial reaction product was also frequently observed in the cis saccules of the Golgi apparatus. These findings suggest that glucose-6-phosphatase may play different functional roles in hormone synthesis within different types of anterior pituitary cells.

The purpose of this study was to characterize the ultrastructural and enzyme-histochemical features of Hofbauer cells in the placental villi of second-trimester abortions. Hofbauer cells in the abortive placenta had more numerous cytoplasmic processes and more prominent phagosomes as compared to gestational age-matched normal control placenta. Acid phosphatase activity was demonstrated in both the lysosomes and on phagosomal membranes of these cells. Our results indicate that Hofbauer cells in second-trimester abortions are activated or stimulated in situ. These activated Hofbauer cells might play important roles in the pathophysiology of preterm parturition.

A cytochemical study of glucose-6-phosphatase (G-6-Pase) was made to see the changes in mucopolysaccharide metabolism in the synovial membrane during osteoarthritis, because the tissue is thought to play an important role in synovial fluid production by providing lubrication to the wear-and-tear surface of the synovial cartilage in osteoarthritis. In normal and osteoarthritic synovial membranes, a high G-6-Pase activity was clearly demonstrated in the cisternal spaces of the rough endoplasmic reticula, especially in the B cells of the thickened synovial membrane. The existence of an AB cells intermediate in character between A and B, was noted in having a high G-6-Pase activity in the accumulated rough endoplasmic reticula in the cytoplasm as B cells do located at the deeper part of the membrane, but also whose cell surface extends to the top of the membrane surface reaching into the synovial cavity, and protrude microvilli of a ruffled border indicating a high phagocytotic activity as A cells do. The existence of the AB cell is clearly one of intermediate structure and function, contributive to the compensatory and regenerative action of the synovial membrane.

Cyclic nucleotide metabolizing enzymes were studied cytochemically to estimate the changes in these enzymes in the synovial membrane, because one of the functions of this tissue is estimated to clean up the debris from the synovial fluid to facilitate lubrication in the knee joints by filtering up small fragments of cartilage which have been formed as the result of wear and tear from the articular cartilage in osteoarthritis. Since the cyclic nucleotides are assumed to regulate the biological activity of phagocytosis, it may be reasonable to expect changes in this activity during the disability of osteoarthritis. In normal and osteoarthritic synovial membranes, a high adenylate cyclase (ACase) activity was clearly observed in the A cells at the surface of the synovial membrane, positive on the plasma membranes of the microvilli and on the ruffled borders as well as in pinocytotic vesicles underneath. The activity of guanylate cyclase (GCase) was observed to be rather weak; however, a strong 5′-nucleotidase (5′-N) activity was evidenced, indicating the existence of a regulatory function with the cyclic nucleotides in the phagocytotic and pinocytotic activities during osteoarthritis.

We present a novel freeze-fracture cytochemistry method based upon enzyme cytochemistry. By this method, freeze-fractured membranes are labeled with cerium as an enzyme cytochemical marker on replicas. The cerium capture method was suitable for freeze-fracture enzyme cytochemistry because the cerium phosphate reaction product is stable after replica-cleaning. As a model system, acid phosphatase, which is a well-known lysosomal marker, was detected on freeze-fractured membranes of lysosomes in the proximal tubular epithelium of the rat kidney. This technique should be a useful addition for analyzing the ultrastructure of biological membranes.

Our objective was to evaluate a method that incorporates a freezing and thawing process to detect cytochrome c oxidase in normal mitochondria in myogenic cell lines in monolayer culture, without the loss of enzyme activity. We used electron microscopy to detect and investigate cytochrome c oxidase activity. When cell preparations were subjected to a freezing and thawing process after being fixed, 91.4% of mitochondria were positively stained. No positive staining was obtained in cells treated by conventional procedures without the freezing process. This method facilitates the positive staining of mitochondria and should be useful in analyzing pathological conditions of mitochondria in cultured cells.

It is of increasing interest to observe the precise localization of cyclic 3′, 5′-nucleotide phosphodiesterase (PDEase) activity in rod outer segments at the ultrastructural level. The cytochemical method of PDEase localization at the ultrastructural level was developed by Florendo et al. However, there were two major difficulties in the use of snake venom as an exogenous 5′-Nucleotidase (5′-Nase): 1) the marked ultrastructural damage due to snake venom, and 2) the inadequate penetration of exogenous 5′-Nase into tissues. Therefore, two improvements for the demonstration of PDEase activity were carried out by the case of: 1) a purified 5′-Nase as exogenous 5′-Nase for a better preservation of cell morphology, and 2) 40μm sections made with the use of a freezing microtome for a better penetration of 5′-Nase into the tissues.PDEase activity using purified 5′-Nase was observed along the disc in the rod outer segment, and no retinal detachment was observed between the outer segments and pigment epithelium, the ultrastructure being excellently preserved, as compared with the use of snake venom. By applying this improved method to freeze-substitution technique, the catalytic site of PDEase was localized on the cytoplasmic surface of the disc membranes. This new technique would be useful in detecting the precise localization of PDEase activity.

Pattern recognition receptors, which include the toll-like receptors (TLRs), are considered to play an important role in the response against lipopolysaccharide (LPS). In this study, we performed a reverse transcriptase/polymerase chain reaction (RT-PCR) study, Western analysis, immunohistochemical staining, and RT-PCR-amplified in situ hybridization of TLR2 and TLR4 in the case of LPS-induced lung injury. The expression of TLR2 and TLR4 increased in the lung rapidly after LPS inhalation and peaked at 24 h, followed by a gradual decrease. TLR2 and TLR4 expression was observed on the bronchial epithelium and tissue macrophages. In the early hours after inhalation of fluorescein-isothiocyanate (FITC)-labeled LPS, LPS was detected mainly on the bronchial epithelium and on a few of tissue macrophages. One day after inhalation, the LPS signals disappeared in the lungs of the mice, except for a few alveolar macrophages. The expression of TLR2, TLR4, and CD14 was coincident with the signals of FITC-labeled LPS. Instillation of liposome-encapsulated dichloromethylene diphosphonate induced a significant decrease in alveolar macrophages. In the macrophage-depleted mice, however, expression of TLR2 and TLR4 mRNA or protein was slightly suppressed in the lung after LPS inhalation. These data suggest that the bronchial epithelium and macrophages play crucial roles in LPS-induced lung injury through TLR2 and TLR4.

We identified five cDNA clones of the Six gene family which are expressed in retina. They are Six2, Six3 alpha and Six3 beta (which are derived from alternative splicing forms), Six5, and AREC3/Six4. All of these Six family genes possess extensive sequence similarity among each other in the so-homologous region (Six domain and homeodomain) but differ greatly in structure in some other regions. The amino acid sequence similarity of the so-homologous region to the previously identified AREC3/Six4 is 70.1% for Six2, 57.3% for Six3 alpha and Six3 beta, and 70.3% for Six5. The expression of these genes was observed in inner and outer nuclear layer, ganglion cell layer, and pigment epithelium of mouse retina by in situ hybridization. The so-homologous region of each Six family protein has specific DNA binding activity. Six5 and Six2 bind to the same sequence as does AREC3/Six4, while Six3 does not. These observations suggest that some of the Six family genes can regulate the same target genes.

In matured chick retina, alkaline phosphatase activity is specifically localized in the outer plexiform layer and in the horizontal and Müller cells. In developing chick retina, the activity is recognized in growing neurites from horizontal cells, when synaptogenesis begins in the outer plexiform layer. Using levamisole, a potent inhibitor of alkaline phosphatase, on chick retina in vivo and in vitro, the enzyme was shown to play a significant role in retinal cell differentiation. 5'-Nucleotidase is used as a marker for the rod photoreceptors. It became apparent that the 'displaced' rod cells are localized in the inner nuclear layer of postnatal retina. High activity of glucose-6-phosphatase is confirmed in the cisternae of the smooth and rough endoplasmic reticulum, together with the space of the perinuclear envelope in the pigment epithelium of rat. The process of disc membrane recycling in the rod outer segment was investigated cytochemically to reveal sequential changes in lysosomal digestion both by conventional enzyme cytochemistry and by high voltage electron microscopy. With conventional enzyme histochemistry as well as with rapid freeze substitution enzyme cytochemistry, all enzyme for cGMP metabolism were observed to be on the cytoplasmic side of the disc membranes.

Dissociated cerebella of newborn rats were cultured in vitro as a monolayer for a long period, and ependymal cell differentiation was investigated by scanning and transmission electron microscopy. Cells with actively beating cilia were recognized on the 3rd day, and gradually increased in number thereafter. At nine months of culturing, a number of areas appeared, some of which consisted of several hundreds of ciliated cells. Cultured ependymal cells showed a striking resemblance in their fine structure to the intact ependyma. They were always found to grow upon the feltwork of astrocytic processes which served as a subependymal structure, as observed in the intact ependyma. It is possible that astrocytes may play some role in ependymal differentiation. Cultured ependymal cells are helpful in understanding the control mechanism for the metachronal cilial movement.

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.

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.

The ultracytochemical localizations of adenosine nucleotidase activities were investigated in the human term placenta. The enzymes studied were as follows: 5′-nucleotidase (5′-N), Ca++-activated adenosine triphosphatase (Ca++ ATPase), Mg++-activated adenosine triphosphatase (Mg++ ATPase) and nucleotide diphosphatase (NDPase). 5′-N activity was demonstrated using not only the lead nitrate method of Wachstein and Meisel but also the recently developed cerium and neodymium method as well.The reaction products for 5′-N activity were found on the external surface of the microvillous plasma membrane of the syncytiotrophoblast. Alpha-beta-methylene adenosine diphosphate (AOPCP), at a concentration of 2.0mM, effectively inhibited 5′-N activity. Ca++ATPase and Mg++ATPase activity were observed strongly on the microvillous membrane of the syncytiotrophoblast and weakly on the basal plasma membrane of the syncytiotrophoblast. NDPase, using ADP as a substrate, was localized on the microvillous membrane.These observations suggest that the syncytiotrophoblast is active in the nucleotide metabolism and that microvillous surface of the syncytium may play an important role in regulating the feto-placental-maternal microcirculation in the human term placenta.

Photoreceptor cell differentiation in the rat retina was studied in vivo and in vitro, using an immunohistochemical method to demonstrate opsin-like immunoreactivity. Cells in a dissociated monolayer culture expressed some properties characteristic of rat rod cells developing in vivo, including a ciliary structure and opsin-like immunoreactivity. Immunoblot analysis revealed that cultured retinal cells synthesize a polypeptide with the same molecular weight as that synthesized by the intact retina. Although the outer segment (OS) was not present in the culture, immunoreactive cells possessed a ciliary structure. Opsin-like immunoreactivity was found on the plasma membrane, including the cilia. The neuritic extensions were also intensely stained. In mature rod cells of the intact rat retina, opsin was detected only on the OS but, during development, it was found both in the somatic region of the rod cells and on the differentiating OS. During maturation of rod cells opsin immunoreactivity seemed to shift to the OS from other locations. However, some "displaced" photoreceptor cells, found in the inner nuclear layer and extending fibers bipolarly, retained immunoreactivity throughout their structure. The absence of polarized distribution of opsin in these cells is considered to be due to an abnormal environment, which may also be the case with cultured retinal cells. The present culture conditions will offer a useful model system to understand the cellular mechanism of the hereditary retinal dystrophy of rodent animals in which photoreceptor cells selectively degenerate.

Normal adult albino and Sprague-Dawley rats, under intraperitoneal Nembutal anesthesia, were used to demonstrate enzymatic activity in the choroid plexus and ventricular ependyma. The brain tissues were perfused or immersed with cold 2% glutaraldehyde and 8% sucrose in 0.1 M cacodylate buffer (pH 7.2–7.4) for 30 min and washed overnight in the same buffer solution. The choroid plexus (lateral and fourth ventricles) and ventricular ependyma (lateral ventricle) were trimmed from the fixed and washed brain tissues, which were frozen and sectioned. For histo- and cytochemical study, the sections were immersed in the following incubation media: for Na+, K+-ATPase (ouabain-sensitive, K+-dependent,p-nitrophenylphosphatase:p-NPPase) according to the one-step method of Mayahara et al. (1978); for Mg2+-ATPase, Wachstein-Meisel's incubation medium (1957); for adenylate cyclase (AC), following Araki and Saito's lead citrate method (1979). The cytochemical findings gave the following results. In the choroid plexus, the ouabain-sensitive electron-dense reaction products of Na+, K+-ATPase (p-NPPase) were strongly positive in the microvilli and along the inner surface of microvilli, without showing any Mg2+-ATPase and AC activities, and all three enzymatic activities were positive along the basal plasmalemmas and negative along the lateral and apical (not including microvilli) plasmalemmas. In the ventricular ependyma, Na+, K+-ATPase (p-NPPase) activity was not found, and the reaction product of AC was observed on the apical plasmalemmas and those of Mg2+-ATPase along the basal plasmalemmas. These cytochemical findings are helpful in understanding the regulation of cerebrospinal fluid production through Na+, K+-ATPase (p-NPPase) and cyclic AMP (AC).

The cholinesterase activity of the Vater-Pacini corpuscle in the periosteum of mouse fibula was histochemically studied by light and electron microscopy. The enzyme activity was confined to the inner core of the corpuscle. It was revealed that the reaction product was found in the perineural spaces between the nerve ending and apposing inner core lamellae, and also in the interlamellar spaces. The reaction product was also found in the nuclear envelope and in the cisterns of the rough endoplasmic reticulum of the inner core cells. The enzyme activity was totally suppressed by eserine 10-4 M and by iso-OMPA 10-4 M. No enzyme activity was demonstrated in the outer core and capsule. The nerve ending showed no distinct enzyme activity. These findings indicate that the mouse Pacini corpuscle has non-specific cholinesteraase activity in its inner core, and that the enzyme may be synthesized in the inner core cells and released into the perineural and interlamellar spaces.

To determine the ultracytochemical localization of Na+, K+-ATPase activity on normal rat choroidal epithelium, K+ dependent-p-nitrophenylphosphatase activity was studied by using the one-step lead citrate method devised by Mayahara et al. (1978).An electron dense reaction product was found to be abundant inside the microvilli and positively along the outside of the basal plasmalemmas of the choroidal epithelium. No reaction product was seen on the apical and lateral ones. In the cytoplasm, the electron dense precipitates were also observed in the endoplasmic reticulum and nuclear membrane. Ouabain (10-3-10-4M) effectively inhibited the activities in the microvilli and basal plasmalemmas. Alkaline phosphatase reaction stronger than that of Mg2+-ATPase was observed mainly on the outside of the basal plasmalemmas, but the activities of these two enzymes were not found in the microvilli.

The modified KUSHIDA and FUJITA method (1967) for in situ PTA staining was applied to the kidney, heart, spleen and brain of the normal adult albino rat. In general, intracellular membranes such as plasma membranes, sarcolemma, membranes constituting junctional areas (synapses, macula adherens, macula occludens etc.), mitochondrial membranes, limiting membranes of lysosomes, membranes of the endoplasmic reticulum including the nuclear envelope were in the negative image, while the mitochondrial matrix, myofilaments, substances in the Z line, the matrix of lysosomes, and nucleoproteins such as ribosomes, chromatin and perichromatin substance were in the positive image. Substances in the cisternae of the endoplasmic reticulum and the basement membrane were also stained moderately positive. Structural similarities observed among the erythrocytic subsurface PTA-positive zone, the subpostsynaptolemmal PTA-positive zone and the macula adherens (grouped as “subplasmalemmal PTA-positive zone”) were suggested in relation to their possible role in ionic movement.The electron lucent layer of membranes seems to correspond to the classical osmiophilic layer. The membrane showed, however, a triple-layered unit membrane-like structure following poststaining with uranyl acetate and/or lead citrate. The PTA-negative electron lucent leaflet of the membrane may be composed of substances with non-polar groups or with a very low concentration of polar groups. Some doubts on the ROBERTSON's unit membrane theory based on the DAVSON-DANIELLI model were expressed.It can be concluded in the present study that ultracytochemistry sine osmium tetroxide is promising for some specific purposes in electron microscopy.

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. Muller 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 Muller 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.

ATPase and alkaline phosphatase (ALPase) activities were histochemically examined in the sensory corpuscle of mouse digital pads at light and electron microscopic levels. ATPase activity was found on the plasma membranes of the lamellar cells. Moderate activity was present in the caveolae, but little activity could be demonstrated in the other portions of the plasma membrane. ALPase activity was found in the caveolae but not on other portions of the plasma membranes of the lamellar cells. ALPase activity was also observed on the nerve terminals. Neither ATPase nor ALPase activity was found on the pre-terminal portions of the nerve fibres. These findings indicate that the caveolae of the lamellar cells may be involved in a transport function and that the plasma membrane of the nerve terminals may have some permeability properties different from those of the pre-terminal portions of nerve fibres.

In order to study cell differentiation and morphogenesis of neural retina, ultracytochemical examination for acetylcholine esterase (AChE) was carried out on neural retinal cells from 6-day-old chick embryos cultured in monolayer for 20 days. AChE is a suitable marker for identifying cell specificity and structure of cultured neural retinal cells, because its specific localization in the intact chick neural retina has been established. After about 2 weeks of culturing a number of cell aggregates formed on the monolayer sheet of glial cells, in which cell bodies were generally located on the periphery regions while their cellular processes were in the center, forming neuropil structures. Among such peripherally located cells presumptive ganglion, amacrine, bipolar, and photoreceptor cells could be distinguished. In the neuropil structures, some cellular processes had typical ribbon synapses indicating that these structures correspond to the plexiform layers of the retina. We could also classify the neuropils into two types of both from the AChE activity and from the structure of the nerve terminals. These findings indicate that our cell culture system can be used for the study of cell differentiation and histogenesis of retinal cells.

Ultracytochemical localizations of cyclic nucleotide-metabolizing enzymes, namely adenylate cyclase (AC), guanylate cyclase (GC) and cyclic 3′,5′-nucleotide phosphodiesterase (PDE), have been demonstrated in the human term placenta. AC activity was found positive on the basal plasma membrane of the syncytiotrophoblast and on the pinocytotic vesicle of the fetal capillary endothelial cell. GC activity was observed to be strong on the plasma membrane of the microvilli of the syncytiotrophoblast. The cAMP PDE activity was shown positive both on the basal plasma membrane and on the microvillous membrane, while cGMP PDE activity was exclusively confined to the microvilli of the syncytiotrophoblast. These observations suggest that the syncytiotrophoblast plays an important role in the cyclic nucleotide metabolism in the human term placenta and that there might be significant functional differences between its basal plasma membrane and its microvillous membrane.

The tissue distribution of enzymatic activities in intestinal metaplasia stomachs exhibiting chronic gastritis was compared histochemically with that of the small intestine in man.

The activity of thiamine pyrophosphatase (TPPase) in the normal rat intestines (jejumun, ileum and colon) was studied. The TPPase activity was strongly positive in the Golgi complex (lamellated cisternae, vesicles and vacuoles) in both the absorptive cells and the goblet cells. Among colonic goblet cells, the Golgi complex of the young maturing goblet cells revealed higher enzymatic activity than that of the well-matured goblet cells containing numerous mucigen droplets. The probable participaton of TPPase with the mechanism of the cyclic secretion of goblet cells through the Golgi complex was postulated.The enzymatic activity was also observed in the plasm membranes covering microvilli, interspaces between lateral cell surfaces forming plications, the nuclear envelope, the rough-surfaced endoplasmic reticulum and occasionally lysosomes in the absorptive cells. The pinocytotic vesicles seen in the vascular endothelium and the smooth muscle cell and the axon-Schwann interfaces revealed the positive enzymatic activity also.