Intense Reaction Product Research Articles

Three cases of placental mesenchymal dysplasia in our hospital

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and β, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl2 precipitation and centrifugation. Vesicles were enriched 11.3 ± 0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system β activity in mouse placental vesicles, measured as Na+-dependent uptake of 14C-methylaminoisobutyric acid (MeAIB) and 3H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system β activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

Isolation of Plasma Membrane Vesicles from Mouse Placenta at Term and Measurement of System A and System β Amino Acid Transporter Activity

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and β, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl2 precipitation and centrifugation. Vesicles were enriched 11.3±0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system β activity in mouse placental vesicles, measured as Na+-dependent uptake of 14C-methylaminoisobutyric acid (MeAIB) and 3H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system β activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

Enzyme histochemical localisation of alkaline phosphatase activity in osteogenesis imperfecta bone and growth plate: A preliminary study

At the ultrastructural level alkaline phosphatase has been studied in calcifying cartilage but not in bone. The aim of this study was to assess if there is an osteoblast dysfunction in Osteogenesis Imperfecta (OI) with respect to alkaline phosphatase activity. Specimens from three OI type II foetal femoral bones, two OI type II growth plates, one normal foetal femoral bone and growth plate, one OI type III femoral bone specimen and one normal juvenile bone specimens were examined using modified lead nitrate method to identify alkaline phosphatase reactivity. The electron dense reaction product (indicative of the presence of alkaline phosphatase) was demonstrable on the cell membrane of the osteoblasts, as focal concentrations in the collagen osteoid and on the mineralisation front of normal bone. In normal bone the intensity of the reaction seemed to be stronger than in OI bone and appeared as a continuous black line along the osteoblast cell membranes. In OI bone the reaction product only appeared as a few electron dense beads along the osteoblast cell membrane. There appeared to be reduced and diffuse reaction product on OI osteoblasts, thus implying either a reduced level and/or altered activity of alkaline phosphatase and hence a dysfunction of osteoblasts. This confirms the findings of the previous report of the impaired activity of alkaline phosphatase in OI osteoblasts. Even in the OI growth plate, hypertrophic chondrocytes showed less intense reaction product than the chondrocytes in the normal growth plate. The normal human growth plates used in this study showed a similar pattern, but in the OI growth plate even the hypertrophic zone, where the alkaline phosphatase activity is reported to be high, showed less intense reaction product. Biochemical reports indicate that alkaline phosphatase levels are normal in cultured OI cell lines, yet ultrastructural histochemical observations reported here, show reduced enzyme localisation and this may suggest reduced amounts of protein or reduced activity at the tissue level.

Cellular localization of tissue inhibitors of metalloproteinases in the rat ovary throughout pseudopregnancy.

The present study determined the ovarian cellular localization of the mRNA for the tissue inhibitors of metalloproteinases (TIMPs) during pseudopregnancy in the rat. Pseudopregnancy was induced by eCG/hCG stimulation. At Day 1 of pseudopregnancy, intense reaction product for TIMP-1 mRNA was observed surrounding the developing corpus luteum (CL), with less intense expression present in granulosa-lutein cells. With continued luteal development, the TIMP-1 mRNA encircling the CL was lost, although low levels of expression were found within the CL. For TIMP-2 mRNA, intense reaction product was observed surrounding the developing CL but, unlike TIMP-1, was present in granulosa-lutein cells, with high levels near the center of the CL. The localization pattern of TIMP-2 mRNA was unchanged through the latter stages of pseudopregnancy. TIMP-3 mRNA expression was strikingly different from the other TIMPs. At Day 1 of pseudopregnancy, intense reaction product for TIMP-3 mRNA was observed in granulosa-lutein cells of certain developing CL, whereas adjacent follicles did not express TIMP-3 mRNA. With continued luteal development, there was a homogenous, intense localization of TIMP-3 mRNA throughout the CL, which was unchanged during pseudopregnancy. To understand the induction of TIMP-3 mRNA in the developing CL, a series of experiments was performed to compare markers of follicular maturity with the presence of TIMP-3 mRNA. TIMP-3 mRNA appears to be switched on in granulosa cells of follicles destined to ovulate. The distinct pattern of expression of the three TIMPs suggests that each inhibitor may regulate either the site and extent of proteolytic action or specific matrix metalloproteinases at different periods of the luteal life span.

Confocal laser scanning and microscopic study of tartrate-resistant acid phosphatase-positive cells in the dental follicle during early morphogenesis of mouse embryonic molar teeth

Tartrate-resistant acid phosphatase (TRAPase)-positive cells were examined during the early morphogenesis of mouse mandibular first molar teeth, using a simultaneous azo dye-coupling technique and confocal laser scanning microscopy (CLSM). CLSM of paraffin-embedded sections revealed that cells exhibiting TRAPase fluorescence appeared along the marginal region of the condensed dental mesenchyme, not yet the dental follicle, at embryonic day 12 (E12). The follicle was recognized histologically as a population of fibroblastic cells in tooth organs at E14, and TRAPase-positive cells were localized among the follicular cells. Light-microscopic observations of microsliced thick sections showed the overall morphology of the TRAPase-positive cells. A few positive cells of E13 tooth organs were oval, and the intense reaction products of TRAPase revealed complex processes on positive cells in E14 tooth organs. Three-dimensional fluorescent images of TRAPase-positive cells using microsliced sections and CLSM showed their unique morphology of long processes with small spine-like projections at E14. Thus, TRAPase-positive cells appeared in the region of the prospective follicle at the bud stage (E12 and 13) before the formation of the follicle proper. In addition, the formation of the follicle and the appearance of TRAPase-positive cells with unique morphology were correlated in their developmental stage (E14). The findings suggest that the TRAPase-positive cells may be involved in the formation of the dental follicle in early tooth morphogenesis.

Intracellular distribution of adenylate cyclase in human cardiocytes determined by electron microscopic cytochemistry

Subcellular localization of adenylate cyclase (AC) in human cardiocytes was studied by electron microscopic cytochemistry using ventricular biopsies from various diseased hearts. In addition to the weak enzyme activity on the sarcolemma, the intense reaction products of AC were demonstrated within distinctive morphologic components of sarcoplasmic reticulum, nuclear envelope, and other internal membranes such as parallel lamellar structures and interlaced tubular structures in the perinuclear regions and stacked membranous structures beneath sarcolemma in cardiocytes. The distribution and intensity of cytochemical activity within different organelles was variable among biopsy cases. The reaction products of AC cytochemistry within the sarcoplasmic reticulum could be related to signal transduction targeting Ca2+ handling by the organella. Cytochemical activity within the nuclear envelope and perinuclear internal membranes possibly reflects AC participation in a signal function to regulate nuclear activity, such as gene expression. Cytochemical distribution of the enzyme in membranous structures beneath the sarcolemma is most likely related to hormone receptors and the linked activity of AC. The subcellular distribution of AC on various internal membrane structures in cardiocytes may reflect compartmentalization of the enzyme at individual intracellular sites to regulate a preferential specific signal function among multiple potential signal transductions by a cascade of AC, cyclic AMP, and cyclic AMP-dependent protein kinase. Alternatively, subcellular localization of the reaction products may reflect local enzyme synthesis or represent sites of enzyme transport, e.g., to terminal localization beneath the sarcolemma. Microsc. Res. Tech. 40:479–487, 1998. © 1998 Wiley-Liss, Inc.

Intracellular distribution of adenylate cyclase in human cardiocytes determined by electron microscopic cytochemistry.

Subcellular localization of adenylate cyclase (AC) in human cardiocytes was studied by electron microscopic cytochemistry using ventricular biopsies from various diseased hearts. In addition to the weak enzyme activity on the sarcolemma, the intense reaction products of AC were demonstrated within distinctive morphologic components of sarcoplasmic reticulum, nuclear envelope, and other internal membranes such as parallel lamellar structures and interlaced tubular structures in the perinuclear regions and stacked membranous structures beneath sarcolemma in cardiocytes. The distribution and intensity of cytochemical activity within different organelles was variable among biopsy cases. The reaction products of AC cytochemistry within the sarcoplasmic reticulum could be related to signal transduction targeting Ca2+ handling by the organella. Cytochemical activity within the nuclear envelope and perinuclear internal membranes possibly reflects AC participation in a signal function to regulate nuclear activity, such as gene expression. Cytochemical distribution of the enzyme in membranous structures beneath the sarcolemma is most likely related to hormone receptors and the linked activity of AC. The subcellular distribution of AC on various internal membrane structures in cardiocytes may reflect compartmentalization of the enzyme at individual intracellular sites to regulate a preferential specific signal function among multiple potential signal transductions by a cascade of AC, cyclic AMP, and cyclic AMP-dependent protein kinase. Alternatively, subcellular localization of the reaction products may reflect local enzyme synthesis or represent sites of enzyme transport, e.g., to terminal localization beneath the sarcolemma.

Das Reaktionsprodukt von Phenylalanin und Ninhydrin: Eine Strukturrevision. Protonentransfer in einemN-unsubstituierten, hochstabilisierten Azomethin-Ylid

The Reaction Product of Phenylalanin and Ninhydrin: Revision of Its Structure. Proton Transfer in an N-Unsubstituted, Highly Stabilized Azomethinylide The formerly proposed structures 6 and 7 for the intense colored reaction product of ninhydrin and phenylalanin are revised in favour of 5a, for which four other tautomeric forms 5b–5e have to be taken into account. Representing a highly stabilized N-unsubstituted azomethinylide 5a can be trapped with N-phenylmaleimide as dipolarophile; 1,3-dipolar cycloaddition and subsequent double bond shift leads to two diastereomeric products 15a and 15b. UV spectrometric data and dynamic 1H NMR measurements reveal an intramolecular proton transfer by hydrogen bonding between 5a and 5b dominating the tautomeric equilibrium.

Cyclic 3',5'-nucleotide phosphodiesterase: cytochemical localization in photoreceptor cells of the fly Calliphora erythrocephala.

The distribution of cyclic 3',5'-nucleotide phosphodiesterase activity was determined in photoreceptor cells of the fly Calliphora erythrocephala. With cAMP as substrate, staining was most intense within the phototransducing region of these cells, the rhabdomeral microvilli and also in the extracellular space surrounding the microvilli and in the mitochondria. With cGMP as substrate, the intensity within the rhabdomeres was less marked, while their extracellular surroundings were stained heavily. Thus, compared to cGMP, cAMP is the better substrate for the phosphodiesterase in the rhabdomeres of the fly. For comparison, the same cytochemical method was used to localize the well-known phosphodiesterase activity in retinal tissue of the mouse. Under the same conditions as used for fly photoreceptors, a very intense reaction product was obtained in rod outer segments. With regard to the conflicting reports concerning the light-stimulated changes of cyclic nucleotides in invertebrate photoreceptor cells, the results presented here further argue for an important role of a cyclic nucleotide in the process of phototransduction of invertebrates.

Immunocytochemical localization of annexin V (CaBP33), a Ca(2+)-dependent phospholipid- and membrane-binding protein, in the rat nervous system and skeletal muscles and in the porcine heart.

We investigated the ultrastructural localization of annexin V a Ca(2+)-dependent phospholipid- and membrane-binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca(2+)-dependent binding of the protein to brain and heart membranes, and on existence of both EGTA- and Triton X-100-extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells.

Immunocytochemical localization of sulfated glycoprotein-1 (SGP-1) and identification of its transcripts in epithelial cells of the extratesticular duct system of the rat.

The localization of sulfated glycoprotein-1 (SGP-1) in the extratesticular duct system was analyzed using an affinity purified antibody raised against the protein in conjunction with light (LM) and electron (EM) microscope immunocytochemistry. In the LM an intense immunoperoxidase reaction product was observed over the cytoplasm of Sertoli cells as well as over the tails of late spermatids. The rete epithelial cells and nonciliated cells of the efferent ducts also showed an intense uniform reaction over their entire cytoplasm. In the EM, immunogold labeling was noted over the entire endocytic apparatus of these cells including coated pits, endosomes, multivesicular bodies, and secondary lysosomes. Since there was no labeling of the luminal contents including sperm along the epididymis, it was concluded that the Sertoli-derived SGP-1 must dissociate from the sperm and be taken up by epithelial cells at the level of the rete testis and efferent ducts. In all regions of the epididymis, except the cauda, the principal cells showed, in the LM, an intense reaction over bodies of various shapes and sizes in their supranuclear region; this corresponded in the EM to a strong immunogold labeling of secondary lysosomes. No labeling was noted, however, over coated pits, endosomes, or pale multivesicular bodies, suggesting that SGP-1 was not being endocytosed from the lumen. Similar observations were noted for the epithelial clear cells along the entire epididymis. In the cauda epididymidis, principal cells presented a weak immunolabeling of their secondary lysosomes. Northern blot analysis revealed a strong 2.6 Kb band corresponding to the mRNA of SGP-1 in the efferent ducts and all regions of the epididymis with the exception of the cauda. Coincident with the mRNA expression of SGP-1 it was found that small clusters of gold particles representing anti SGP-1, presumably membrane bound, were associated with the Golgi apparatus as well as in close proximity to secondary lysosomes. There was, however, no evidence for the secretion of SGP-1 into the lumen. These results suggest that SGP-1 is synthesized by the epithelial cells of the male duct system and ferried by small vesicles derived from the Golgi apparatus to secondary lysosomes. Because SGP-1 has recently been shown to have substantial sequence similarity to prosaposin, it may be speculated that SGP-1 is instrumental in the degradation of membrane glycolipids present within secondary lysosomes of epithelial cells of the extratesticular duct system.

Subcellular localization of insulin-like growth factor I (IGF-I) in Purkinje cells of the adult rat: An immunocytochemical study

Insulin-like growth factor I (IFG-I) was localized by light and electron microscopic immunocytochemistry in the adult rat cerebellum. Neurons showing the same cytological characteristics and pattern of synaptic input as Purkinje cells were IGF-I immunoreactive. In these neurons, intense electron-dense reaction product was consistently located in the rough endoplasmic reticulum and, occasionally, in multivesicular bodies and associated with the nuclear envelope. Thus, the results of this study suggest synthesis IGF-I in Purkinje cells and furthermore provide anatomical support for IGF-I participation in paracrine or autocrine regulatory systems in the brain.

Localization of type IV collagen in the basement membranes of human prostate and lymph nodes by immunoperoxidase and immunoalkaline phosphatase

The object of these studies was to examine the localization of type IV collagen (Coll-IV) in the basement membranes (BM) of epithelial and stromal elements (smooth muscle, nerves, vessels) in normal, hyperplastic, and neoplastic (primary and metastatic) prostate. We also examined the relationship of Coll-IV distribution to the degree of tumor differentiation (Gleason grading system). We compared immunoperoxidase (IP) and immunoalkaline phosphatase (AP) techniques in these studies and in selected samples we also evaluated immunofluorescence (IF) localization of Coll-IV and the effects of tissue fixation and pepsin digestion. We found that IF localization of Coll-IV was intense in unfixed sections. IP and AP reactions were absent in fixed, paraffin-embedded sections but pepsin treatment yielded intense and uniform reaction products in these same preparations. Both the IP and AP techniques showed similar localization of Coll-IV in the BM of normal, hyperplastic, and well-differentiated tumor. In most of the higher-grade tumors Coll-IV localization was reduced and a similar pattern of distribution was observed after IP and AP techniques. However, in some high-grade tumors the IP technique showed good localization but AP did not, and vice versa. Such discrepancies were noted in the BM of the tumor cells, as well as in the BM of the stromal elements and in lymph nodes with metastatic tumor. Thus, our study shows decreased Coll-IV localization in higher-grade tumors and suggests that the use of a single technique (IP or AP) may exaggerate this apparent loss of Coll-IV BM components. The exact cause of these discrepancies is unknown but they must reflect variable losses in the ability of the tumor cells to form BM, degradation or decreased synthesis of BM components by high-grade tumors, or a combination of the above.

Ultracytochemical Demonstration of Ca<sup>2+</sup> -ATPase Activity in the Rat Saphenous Artery and Its Innervated Nerve Terminal

The localization of Ca2(+)-ATPase activity was ultracytochemically investigated in the rat saphenous artery and nerve terminals innervating the saphenous artery using a lead citrate method devised by Ando et al. (1981). Intense reaction products in the saphenous arterial endothelial cells were observed inside the caveolae and vesicles along the luminal and abluminal sides. In addition, Ca2(+)-ATPase activity was observed on the external side of the luminal, abluminal and lateral plasma membrane, and the outer membrane of mitochondria. In the smooth muscle cells, intense Ca2(+)-ATPase activity on the inside of caveolae and vesicles was observed, comparing in intensity with that on the plasma membrane of smooth muscle cells. In the nerve terminals innervating the saphenous artery, Ca2(+)-ATPase activity was demonstrated on the plasma membrane of the nerve terminal-Schwann cell interface, the axolemma of unmyelinated axons and the plasma membrane of Schwann cells. It is suggested from the above ultracytochemical results that Ca2(+)-ATPase activity plays an important role in the contraction and relaxation of the saphenous artery, and in the neurotransmitter release.

Localization of urokinase-type plasminogen activator in human eyes: An immunocytochemical study

We investigated the presence of urokinase-type plasminogen activator (u-PA) in various structures of the human eye by using a biotin-strepavidin complex immunoperoxidase technique with a monoclonal antibody specific for human u-PA. A moderate to intense reaction product was seen in the corneal endothelium, episclera, sclera, iris muscles, lens equator and posterior capsule, peripheral vitreous, epithelium of pars plana, retinal pigment epithelium, axons, uveal fibroblasts, optic nerve fibers, and extraocular muscles. A weak reaction product was seen in the conjunctival and corneal epithelium, the trabecular meshwork, melanocytes and stroma of the iris and choroid, and the posterior layers of the retina. u-PA was not localized in the corneal stroma, conjunctival goblet cells, and certain parts of the lens. Comparison of the present results with those of our previous study (Tripathi, Geanon and Tripathi, 1987, Ophthalmology, 94, 1434-8) on the localization of tissue plasminogen activator (t-PA) in the eye indicate that the distribution of u-PA and t-PA overlaps in many tissues, but differs significantly in others, and that this finding may be related to their complementary and specific roles. As in other body systems, u-PA in ocular tissues is probably involved in processes such as fibrinolysis, wound healing, turnover of intra- and extracellular macromolecules, muscle and neuromuscular regeneration, synapse formation, and maintenance of general neuronal function.

Dynamics of Gastrin‐producing Cells in the Rat after Truncal Vagotomy Evaluation by Double lmmunostaining for Bromodeoxyuridine and Little Gastrin

The mechanism of hyperplasia of gastrin-producing cells (G-cells) in the rat antral mucosa after truncal vagotomy was studied using double immunostaining for bromodeoxyuridine (BrdU) and little gastrin (G17). With single labeling of BrdU, a few G-cells (less than 1%) showed positive immunostaining for BrdU in the nucleus throughout the experimental period in both vagotomized rats and those given a sham operation. The labeled cells in both groups demonstrated a linear increase of BrdU labeling in an identical number of cells for each experimental time-point. The labeling index of the G-cells increased rapidly from day 2 to day 6 and attained a maximum level of 44.0% on day 10 in the vagotomized group after cumulative labeling. Even in this group, however, many G-cells showed no BrdU immunoreactivity throughout the experimental period. These cells did not replicate during the experimental period, but showed an intense reaction product for G17 in their cytoplasm after vagotomy. The present study indicates that the most important factor involved in G-cell hyperplasia observed after truncal vagotomy is the activation of pre-existing G-cells to synthesize and release hormone, together with the rapid maturation of progenitor cells to mature G-cells.

Water channels and water canals in the gill of the freshwater mussel, Ligumia subrostrata: Ultrastructure and histochemistry

AbstractThe apical surface of all epithelial cells of the gills of Ligumia subrostrata either are ciliated or contain microvilli or show both structures. The ciliated cells on the gill filaments direct water through the ostia at the base of each gill filament. All the water passing through the gills and out the exhalent siphon must pass through the narrow water canals and into the central water channels. All of the epithelial cells lining the lateral water canals are mitochondria‐rich. In addition, the central water channel epithelia contain clusters of mitochondria‐rich cells, but many of the epithelial cells are mitochondria‐poor. With the exception of the lateral ciliated cells, the lamellar epithelium of the surface filaments contains relatively mitochondria‐poor cells. The mitochondria‐rich epithelial cells of the water canals and water channel display the most histochemically intense reaction products for carbonic anhydrase and cytochrome oxidase. The mitochondria‐rich cells of the water canal and water channel epithelia may be the principal cells responsible for salt transport in these mussels.

Peroxidase Activity in Benign Nodular Lesions and Carcinoma in the Rat Thyroid Induced by N‐nitrosobis (2‐hydroxypropyI) amine and Phenobarbital

Nodular lesions induced by N-nitrosobis (2-hydroxypropyl) amine (BHP) and phenobarbital (Pb) in F344 rat thyroid were histologically divided into 2 categories, benign lesions and carcinoma. Benign lesions were subclassified into 3 types mainly based on histological characteristics and localization of peroxidase (PO). The type 1 lesion showed localization of PO almost identical with that of the group 1 rat thyroid. Type 2 was characterized by an intense reaction product of PO in the cytoplasm but a weak one on the surface of the microvilli, and type 3 exhibited localization of PO opposite to that of type 2. Carcinoma proliferated in papillary, follicular and poorly differentiated patterns and developed as de novo carcinoma with the highest incidence, although a considerable number of carcinomas coexisted with benign type 3 lesions. A majority of these carcinomas lacked the reaction product for PO. These results suggest that benign nodular lesions, especially types 2 and 3, show hyperactivity of PO synthesis reflecting their response to excessively secreted TSH, whereas carcinoma is autonomous, because of marked inhibition of PO synthesis even in the presence of TSH hypersecretion.

Histochemical localization of 5'-nucleotidase in the lymphatic endothelium.

Histochemical localization of 5′-nucleotidase (5′-Nase) in the lymphatic endothelium of rat stomach was studied by combined light and electron microscopy. Lymphatic capillaries and blood capillaries were distinguished histochemically on cryostat sections using 5′-Nase-alkaline phosphatase double staining.The distribution of lead-demonstrated 5′-Nase activity in the lymphatic capillaries could be determined by comparing the images of the same section as seen by light and backscattered imaging scanning electron microscopy. The specificity of the 5′-Nase reaction was obtained by inhibiting nonspecific alkaline phosphatase by including L-tetramisole in the incubation medium for 5′-Nase activity. The intensity and localization of 5′-Nase activity on the walls of lymphatic capillaries were also determined by the lead-based or cerium-based method. The intense reaction products of 5′-Nase activity were predominantly deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells. The cerium-based method revealed fine and linear reaction products on the plasma membrane.

An Immunohistochemical Study of Cytokeratin and Vimentin in Benign and Malignant Thyroid Lesions

Intermediate filaments in benign and malignant thyroid lesions were immunohistochemically studied using polyclonal and monoclonal anti-cytokeratin (CK), and monoclonal anti-vimentin antibodies. Antigenicity of CK and vimentin was almost completely destroyed during formalin fixation in normal thyroid and all thyroid lesions except for some cases of papillary and squamous cell carcinoma, although the latter showed negative immunostaining with anti-vimentin antibody. In sections fixed with Carnoy's fixative, most cases of papillary carcinoma showed an intense reaction product for polyclonal anti-CK, monoclonal anti-CK-7, CK-19 and anti-vimentin antibodies. The reaction product for anti-CK antibodies was located mainly in the apical cytoplasm and that for anti-vimentin antibody in the basal cytoplasm. However antigenicity was still destroyed by the fixative in many specimens of normal thyroid, benign thyroid lesions and follicular carcinoma. In frozen sections, all specimens showed preserved antigenicity for both antigens with an intense reaction product in papillary carcinoma, but this was weaker in normal thyroid, benign thyroid lesions and follicular carcinoma. Therefore, follicular cells under normal and pathological conditions contain intermediate filaments of CK and vimentin in their cytoplasm and co-expression of the antigens is significantly increased in papillary carcinoma.