Nase Activity Research Articles

АКТИВНОСТЬ ДЕГИДРОГЕНАЗ В НЕФТЕЗАГРЯЗНЕННЫХ ПОЧВАХ КАК ИНСТРУМЕНТ МОНИТОРИНГА ТЕХНОЛОГИЙ БИОРЕМЕДИАЦИИ

The dynamics of dehydrogenase activity in a southern chernozemic oil-contaminated soil during natural attenuation and by using two remediation techniques (stimulation of an indigenous microflora and introduction of active oil-oxidizing Dietzia maris strain AM3) was examined in laboratory experiments. It was shown that dehydroge­ nase activity expediently use to estimate the initial bioremediation processes and for monitoring the remediation process by bioaugmen­ tation.

Expression of CCL21 and 5???-Nase on Pancreatic Lymphatics in Nonobese Diabetic Mice

To obtain better insight into the modulation of lymphatic endothelial cells during the autoimmune process, alterations of the structures and histochemical features in the pancreatic lymphatics were studied in nonobese diabetic (NOD) mice. The expression of secondary lymphoid tissue chemokine (SLC/CCL21) and 5'-nucleotidase (5'-Nase) on pancreatic lymphatics was examined by histochemistry and immunoblot in NOD mice. As insulitis developed, the increased expression of CCL21 and podoplanin on pancreatic lymphatics was consistent with the increased number of cytoplasmic protrusions and vesicles, whereas 5'-Nase activity of lymphatics seemed to become decreased. The expression of CCL21 protein also showed an age-dependent increase in NOD pancreas, even though it was undetectable in normal controls. During the period of severe infiltration, reaction products of CCL21 and podoplanin were detected in the nucleus and cytoplasm of lymphatic endothelial cells. Dendritic cells and T lymphocytes frequently penetrated through the slender walls of lymphatics and adhered to the lymphatic luminal surfaces, precipitating with few 5'-Nase particles. In contrast to wild-type NOD mice, complete Freund adjuvant administration reduced CCL21 expression in NOD pancreas, suppressing the entry of activated dendritic cells into lymphatics. These findings suggest that CCL21 and 5'-Nase may be involved in the interaction between infiltrating cells and lymphatic vessels to induce the functional changes of lymphatic endothelial cells during insulitic and diabetic development.

Lymphatic network and lymphangiogenesis in the gastric wall.

A family of growth factors highly specific for endothelial cells was identified more than 10 years ago, in which the receptor of vascular endothelial growth factor C (VEGFR-3) is implicated in the regulation of lymphatic development and regeneration. Comparative studies on the lymphatic network and lymphangiogenesis have been done mainly using combined 5'-nucleotidase (5'-Nase) enzyme and VEGFR-3 immunohistochemical approaches in adult and fetal gastric walls. Developing lymphatic networks represented fewer blind ends and branches than mature networks in whole-mount preparations. Many circular lymphatic-like structures exhibited VEGFR-3 expression and weak 5'-Nase activity in the early embryonic stage, showing visible morphological properties in the lymphatic endothelium. These newly formed lymphatics showed an obvious accumulation in the submucosa and serosa and a variation in the intensity of VEGFR-3 binding to endothelial cells among samples. A reaction product for anti-VEGFR-3 was found on the luminal surface of endothelial cells and on the membrane of some organelles and intraluminal lymphocytes. These findings indicate that an active proliferating feature of the clustered developing lymphatics may create a favorable environment for their sprouting and growth, which may serve as a functional requirement for lymph drainage in the region.

Demonstration of the Rat Lymphatic Vessels Using Immuohistochemistry and In Situ Hybridization for 5^|^apos;-Nucleotidase

The cellular distribution of 5'-nucleotidase (5'-Nase) was studied in the rat tissues at the levels of protein and mRNA by use of immunohistochemistry and in situ hybridization, and compared with that of reaction productsby enzyme-histochemistry. 5'-Nase activity, immunoreactivity of ecto-5'-Nase (CD73) and hybridization signals for its mRNA were colocalized in the lymphatic vessels including central lacteals of the small intestine, suggesting that 5'-Nase is actually produced in the lymphatic endothelial cells and allocated to their cell membrane as an enzyme to regulate lymph production and flow. Double immunostainig for CD73 and Factor VIII-related antigen clearly showed the distinction and relationship between lymphatics and the blood vessels. The present findings support our view that 5'-Nase is an available marker of lymphatics, and indicate the usefulness of the histochemical methods for 5'-Nase not only for demonstration of lymphatics, but also for examination of the functional roles and dynamics of 5'-Nase in the lymphatic endothelial cells in physiological and pathological conditions.

Influência do pH do meio de cultivo e da turfa no comportamento de estirpes de Bradyrhizobium

A acidez dos solos representa um problema que afeta grandes áreas agrícolas pelo mundo, principalmente nos trópicos, onde fósforo e nitrogênio também são limitantes. No caso do nitrogênio, a fixação biológica torna-se uma das alternativas mais viáveis do ponto de vista ecológico e econômico, por diminuir o uso e o impacto causado pelos fertilizantes nitrogenados. Neste trabalho, foram realizados dois experimentos in vitro e um em casa de vegetação com quatro estirpes de Bradyrhizobium (Br 4406, Br 29, SEMIA 587 e INPA 03-11B), no Departamento de Ciência do Solo (UFLA), de julho de 1998 a julho de 1999, para verificar o efeito de três valores de pH (5,0; 6,0; 6,9) no crescimento destas em meio de cultura YM, na sua simbiose com soja, assim como na sua sobrevivência em inoculantes produzidos com turfa. No primeiro experimento, as estirpes de Bradyrhizobium tiveram um comportamento diferenciado em meio líquido, obtendo melhor desempenho em pH 6,0, tanto em número de unidades formadoras de colônias quanto em produção de exopolissacarídeos. +No segundo experimento, o número de nódulos, a atividade da nitrogenase (Nase), as massas secas de nódulos, raízes e parte aérea de plantas de soja, de modo geral, não foram influenciados pelos valores de pH de cultivo das estirpes no inoculante. Entretanto, a estirpe INPA 03 - 11B mostrou-se efetiva, apresentando número de nódulos e atividade de Nase semelhantes aos de Br 29 e SEMIA 587, que são recomendadas como estirpes inoculantes, devendo, assim, ser indicada para testes de eficiência em campo. No terceiro experimento, com exceção da Br 29, que atingiu maior sobrevivência de células em pH 6,0, as outras estirpes tiveram sobrevivência semelhante neste valor de pH e em pH 6,9. O melhor desempenho das estirpes de Bradyrhizobium em pH 6,0 no meio de cultura e em turfa demonstrou a possibilidade do uso de inoculantes corrigidos para esse valor de pH, como modo de pré-adaptação às condições de acidez dos solos tropicais.

Soil properties and nutrient uptake as influenced by mulching in maize (Zea mays)-based cropping systems

A field experiment was conducted at the ICAR Research Complex for NEH Region, Umiam, Meghalaya (950 m above sea-level) during the rainy (kharif) and winter (rabi) seasons of 201213, to study the effect of soil-moisture conservation (SMC) measures on nutrient uptake and soil fertility in maize (Zea mays L.)-based cropping se- quences. Significantly the highest soil organic carbon (SOC) was recorded under maizeFrench bean (Phaseolus vulgaris L.) (pole type) system (1.72%) followed by maizeblackgram [Vigna mungo (L.) Hepper] (1.70%) at 015 cm depth. In-situ maize stover mulch (MSM) + white hoarypea (Tephrosia candida DC.) @ 10 t/ha (on fresh weight basis) mulch resulted in significantly higher SOC (1.72%), followed by MSM + ragweed (Ambrosia artemisiifolia L.) mulch 10 t/ha (1.69%). The highest soil available N (268.6 kg/ha) and P (15.7 kg/ha) were recorded under maize blackgram and K (277.6 kg/ha) under maizeFrench bean cropping system. The MSM + Tephrosia mulch re- corded significantly higher soil available N, P and K, followed by MSM + Ambrosia. MaizeFrench bean (pole type) cropping system had significantly higher soil microbial biomass carbon (SMBC; 258.55 ?g/g soil) and dehydroge- nase activity (DHA; 37.27 ?g TPF/hr/g) than that under maizefallow. Among the SMC measures, MSM + Tephrosia recorded the maximum SMBC (265.75 ?g/g soil) and DHA (34.27 ?g TPF/hr/g) followed by MSM + Am- brosia. On an average, nutrient (N, P and K) content and uptake in seed and stover of all the rabi crops were the highest under MSM + Tephrosia followed by MSM + Ambrosia system. The highest maize-equivalent yield was ob- tained from maizeFrenchbean (pole type) system under MSM + Tephrosia mulch (9.5 t/ha), followed by MSM + Ambrosia mulch (8.8 t/ha).

Histochemical analysis of lymphatic endothelial cells in lymphostasis.

The ultrastructure of endothelial cells of intestinal lymphatics and the thoracic duct (TD) and the relation to lymphostasis were examined in rats and monkeys. Localization of 5'-nucleotidase (5'-Nase) and endothelial nitric oxide synthase (eNOS) was studied. In normal lymphatic endothelial cells, 5'-Nase reaction product was evenly deposited on the cell surface in vivo and on cultured TD endothelial cells (TDECs), whereas eNOS was evenly distributed throughout the nucleus and cytoplasm. TDECs had a long filamentous process extending towards the subendothelial extracellular matrix but became flat and regular within 30-40 minutes after gastric perfusion with olive oil. According to their electron-density, two types of cells were found in the TD endothelial layer. The cells with low electron-density exhibited stronger 5'-Nase activity. Valves were bicuspid formations and the valvular endothelial surface of the convex side showed weaker 5'-Nase activity than the concave side. During TD blockage-induced lymphostasis in rats, the 5'-Nase product was almost not discernible in the TDECs within 2 weeks. Larger vesicles were found in the endothelial cytoplasm of the ligated TD. Their number decreased after 6-12 weeks. The small intestinal lymphatics in the mucosa and submucosa were dilated, with numerous open intercellular junctions. The endothelial lining appeared to have reduced activities for 5'-Nase and eNOS in 9 of 11 experimental animals. The results indicated that the inability of the open intercellular junctions, normally working as one-way endothelial flap valves, may be a key morphological feature after TD blockage. Reduced eNOS and 5'-Nase may functionally influence contractile activity and transport capability of the lymphatic vessels in the lymphostasis.

Enzyme-histochemical demonstration of lymphatic vessels in the golden hamster periodontium: an attempt to reactivate the 5'-nucleotidase activity with Mg++ ion supply.

Enzyme-histochemical demonstration of lymphatic vessels in the golden hamster periodontium was performed on cryostat sections using the 5'-nucleotidase (5'-Nase) staining method by light microscopy and backscattered electron imaging of scanning electron microscopy. The inhibition of the 5'-Nase activity by decalcification was cancelled by the Mg++ ion supply. The reaction products of 5'-Nase activity were produced on the lymphatic endothelial cells and the tubular structures of lymphatic vessels were seen as highlights by backscattered electron imaging. The invasion of 5'-Nase-positive lymphatic vessels into the alveolar bone from the periodontium was found in the present study.

Expression of purine metabolism-related enzymes by microglial cells in the developing rat brain.

The nucleoside triphosphatase (NTPase), nucleoside diphosphatase (NDPase), 5'-nucleotidase (5'-Nase), and purine nucleoside phosphorylase (PNPase) activity has been examined in the cerebral cortex, subcortical white matter, and hippocampus from embryonic day (E)16 to postnatal day (P)18. Microglia display all four purine-related enzymatic activities, but the expression of these enzymatic activities differed depending on the distinct microglial typologies observed during brain development. We have identified three main morphologic typologies during the process of microglial differentiation: ameboid microglia (parenchymatic precursors), primitive ramified microglia (intermediate forms), and resting microglia (differentiated cells). Ameboid microglia, which were encountered from E16 to P12, displayed the four enzymatic activities. However, some ameboid microglial cells lacked 5'-Nase activity in gray matter, and some were PNPase-negative in both gray and white matter. Primitive ramified microglia were already observed in the embryonic period but mostly distributed during the first 2 postnatal weeks. These cells expressed NTPase, NDPase, 5'-Nase, and PNPase. Similar to ameboid microglia, we found primitive ramified microglia lacking the 5'-Nase and PNPase activities. Resting microglia, which were mostly distinguishable from the third postnatal week, expressed NTPase and NDPase, but they lacked or displayed very low levels of 5'-Nase activity, and only a subpopulation of resting microglia was PNPase-positive. Apart from cells of the microglial lineage, GFAP-positive astrocytes and radial glia cells were also labeled by the PNPase histochemistry. As shown by our results, the differentiation process from cell precursors into mature microglia is accompanied by changes in the expression of purine-related enzymes. We suggest that the enzymatic profile and levels of the different purine-related enzymes may depend not only on the differentiation stage but also on the nature of the cells. The use of purine-related histoenzymatic techniques as a microglial markers and the possible involvement of microglia in the control of extracellular purine levels during development are also discussed.

INHIBITION OF ARACHIDONIC ACID ESTERIFICATION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO OZONE IN VITRO

There is evidence that some lung responses to ozone (O3) exposure are mediated through the altered metabolism of arachidonic acid (AA). Increased concentrations of some AA metabolites such as prostaglandin E2 (PGE2) and prostaglandin F2 (PGF2) are observed in the bronchoalveolar lavage fluid recovered from human subjects exposed to O3. Airway epithelial cells may contribute to this increase in eicosanoid formation. Previous reports have shown increased PGE2 and PGF2 production by O3-exposed air way epithelial cells, believed to be mediated at least in part by increased phospholipase activity. We examined other potential biochemical mechanisms for the increased formation of these two prostaglandins by O3-exposed epithelial cells. Cultured normal human bronchial epithelial (NHBE) cells, prelabeled with 3H-AA, were exposed to air or to 0.1 ppm or1.0 ppm O3 for 60 min in a Transwell cell culture system. NHBE cells produced increased amounts of 3H-PGE2 and 3H-PGF2 in response to 0.1 ppm O3 exposure but not with 1.0 ppm exposure as measured in the conditioned media by high-performance liquidchromatography. Other 3H-AA products, including [3H]-15-hydroxyeicosatetraenoic acid, [3H]-12-heptadecatrienoic, and [3H]aldehydic substances derived from the ozonation of 3H-AA, also were observed in increased amounts. Pulsing of cells with 15 n 3H-AA after 0.1 ppm and 1.0 ppm O3 exposure revealed a decrease in 3H-AA esterification into cellular phospholipids, resulting in an increase in free 3H-AA available for metabolism to prostaglandins. No O3-induced alteration in NHBE cell cyclooxyge nase activity was observed with the 0.1 ppm exposures. Impaired esterification of free AA into cellular phospholipids appears to be a very sensitive target for O3-induced effects on AA metabolism, and may play a role in the increased prostaglandin production observed upon O3 inhalation in vivo.

Demonstration of the intralobular lymphatics in the guinea pig pancreas by an enzyme-histochemical method.

Intralobular lymphatics in the guinea pig pancreas were demonstrated enzyme-histochemically showing the extent, distribution and fine structure by combined light and transmission electron microscopy. 5'-nucleotidase(5'-Nase)-positive lymphatic vessels were present throughout the pancreas. Intralobular lymphatics among the acini were comparatively rare and generally independent of the blood capillaries, pancreatic ducts and acini. These lymphatics revealed the usual structural features, such as typical intercellular junctions and very tenuous vascular walls without continuous basal laminae. Fine precipitates of the cerium-based reaction product for 5'-Nase activity were found to be associated with cell membranes of the lymphatic endothelium and pinocytotic vesicles. Lymphatics were not closely related to the endocrine islets, although alkaline phosphatase(ALPase)-positive blood capillaries were well developed. Collecting lymphatic vessels with valves with weaker 5'-Nase activity were also detected in the interlobular connective tissue. ALPase activity, absent in the lymphatics, was positive in the blood capillaries, suggesting that it is also a useful way demonstrating, histochemically, the blood capillaries in the guinea pig pancreas.

Enzyme-Histochemical Study on Postnatal Development of Rat Stomach Lymphatic Vessels

Postnatal development of rat gastric lymphatics was studied by an enzyme-histochemical method to elucidate the morphological changes of lymphatics and their relationship to maturation and function, especially in the glandular portion. The significant features of 5′-Nase-positive lymphatics in distribution and structure were examined in different stages (within 24 hr, 4–21 days, and 2 months). Lymphatics in the greater curvature and anterior wall grew much slower than those in the lesser curvature and posterior wall of the stomach in newborn and infant rats. Lymphatic islands isolated from the primary lymphatic networks in the submucosa and subserosa underwent a morphological change during this early period. This is considered one of the basic steps in lymphatic development. Occurrence of lymphatic networks in the deep lamina propria indicates that development in the gastric wall is well characterized from Day 10. With further growth and modification of lymphatics, the networks in the different layers formed an extensive communication network and many lymphatic valves were found in the submucosa and subserosa. Pinocytotic vesicles, open junctions, and intraendothelial channels were frequently detected in the mucosal and submucosal lymphatic networks of the corpus–antrum and antrum–duodenum divisional zones in the adult rats. These findings suggest that developing lymphatics in the rat stomach may represent rapidly growing tissue not only with high 5′-Nase activity but also with high adaptability for future physiological demands.

Detached retina affects morphologic and biochemical changes in the retina adjacent to bullous retinal detachment in rabbits

PURPOSE. Long-term results, more than 10 years after successful retinal detachment surgery, have shown gradually decreasing visual acuity in some cases. It is unclear if reduced functional recovery postoperatively is caused by anatomic changes or biochemical disorders. To determine the etiology of the reduced visual acuity, we cytochemically examined the changes in the cellular responses of the edges of retinal detachments. METHODS. We histochemically studied the glucose-6-phosphatase (G6P) and 5'-nucleotidase (5'-Nase) activity in the rabbit retina. Experimental rhegmatogenous retinal detachment was produced in a rabbit model after partial vitrectomy, followed by retinal tear formation. RESULTS. Although 5'-Nase activity gradually decreased during the period of detachment, activity was still detectable after 24 weeks. G6P activity increased in the region of the detached neural retina. Around the border of the detached retina, the decrease in 5'-Nase activity extended approximately 140 micrometers into the adjacent attached retina at 2 weeks after detachment and 270 micrometers at 24 weeks. CONCLUSIONS. These observations suggest that some anatomical and biochemical damages may occur in the retina adjacent to bullous retinal detachment and may explain the reduction in postoperative vision in some clinical cases.

Enzyme-histochemical demonstration of the intramural lymphatic network in the monkey jejunum.

The structural organization of the intramural lymphatic networks in the jejunum of the Japanese monkey (Macaca fuscata) was studied by an enzyme-histochemical method using whole-mount preparations of tissue layers. A distinction between the lymphatics and the blood vessels was made by light microscopy using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) double staining. In each tissue specimen, the reaction product for 5'-Nase activity was localized in the lymphatic vessels, whereas that for ALPase activity was restricted to blood vessels. The mucosal layer exhibited an irregular network of tubular lymphatics connecting to the central lymphatics in the villi. The submucosal layer revealed a horizontal mesh-like network consisting of tubulo-saccular lymphatics. A dense maze-like network of lymphatics was distributed throughout the myenteric layer. The lymphatic networks were independent of the blood vessels. The present results indicate the usefulness of the enzyme-histochemistry of whole-mount preparations of the laminar layers for demonstration of the structure and distribution of the lymphatic system in the intestinal wall.

SaRD, A New Protein Isolated from the Extremophile Archaeon Sulfolobus acidocaldarius, Is a Thermostable Ribonuclease with DNA-Binding Properties

We have isolated the thermostable 9 kDa SaRD -protein from S ulfolobus a cidocaldarius which exhibits R Nase activity as well as D NA-binding properties (SaRD). The amino acid composition and the sequence of the 16 N-terminal amino acids show similarities to different RNases as well as to DNA-binding proteins from thermophilic archea. The RNase activity was demonstrated by 5S rRNA degradation, thin layer chromatography and a zymogram. The temperature optimum for the RNase activity is 65°C. The pH optimum ranges from 6.5-7.0. DNA-binding properties were shown by gel-shift assays on agarose gels. In a similar way SaRD mediated protection of DNA against DNase I digestion and Sau3A I restriction could be demonstrated. The melting point (T m) of genomic DNA was raised from 68°C to 90°C by addition of the SaRD-protein. CD spectroscopy indicated that SaRD is very stable near neutral pH and can neither be unfolded by temperatures up to 85°C nor by addition of 8 M urea.

誘発舌癌における毛細リンパ管の酵素組織化学的観察

Assessment of the distribution of lymphatic capillaries in the connective tissue underlying tumors is very important when attempting to explain the mechanism by which cervical lymph node metastases arise from tongue cancer. The left lateral border of the hamster tongue was rubbed with a dental cleanser in the control group. In the experimental group, it was further treated with a 2.0% solution of 9, 10-dimethyl 1, 2 -benzanthracene acetone (DMBA) to induce an infiltrated tongue cancer. Frontal sections of the tongue from each group were stained with 5'-nucleotidase (5'-Nase) and the lymphatic capillaries were examined under a light microscope.In the control group, the lymphatic capillaries were present in all connective tissue papillae. These lymphatic capillaries arose from the lymphatic capillary network in the lamina propria mucosae. In the experimental group, the lymphatic capillaries were also present in the connective tissue underlying the tumor so that the connective tissue structure resembled papillae. Lymphatic capillaries underlying the tumor were significantly thicker in diameter than those in the control group. The 5'-Nase activity was lower in the experimental group than in the control group.It is conceivable that an increase in diameter is necessary so that the lymphatic capillaries can absorb a large quantity of tissue fluid. Lymphatic capillaries underlying the tumor always maintained a plexus. This result suggested that the lymphatic capillary network in the connective tissue underlying the tumor was repeatedly built and destroyed as the tumor invaded the muscle layer. An inverse relationship between 5'-Nase activity and the diameter of the lymphatic capillaries was also suggested by the results of this study.Therefore, 5'-Nase staining was very useful for observing the lymphatic capillaries in the connective tissue underlying infiltrated squamous cell carcinoma of the tongue.

Identification of subcellular targets and sensitive tests of ethanol-induced damage in isolated rat gastric mucosal cells

Background/Aims: The mechanisms of chemically induced injury are poorly understood. The purpose of this study was to develop a rapid method for isolation of gastric mucosal cells (GMCs) and sensitive tests for investigating cell damage and protection. Methods: GMCs were prepared from rat glandular stomach with pronase and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and kept in albumin-containing, albumin-free, or Hank's solutions. Initial cell viability and responsiveness were tested with trypan blue exclusion, and stimulation or inhibition of pepsinogen secretion were tested with carbachol, 16,16-dimethyl prostaglandin E2, or carbachol plus atropine. Plasma membrane damage was assessed by trypan blue exclusion, lactate dehydrogenase or cell protein leakage, mitochondrial integrity by succinic dehydroge nase activity, and nuclear lesions by ethidium bromide DNA fluorescence. Results: Incubation of GMC with ethanol resulted in 50% lethal dose for ethanol values of 12.3%, 7.4%, and 10.9% by trypan blue exclusion; 10.1%, 9.4%, and 9.1% by lactate dehydrogenase leakage; 12.8%, 10.0%, and 10.4% by succinic dehydrogenase; and 14.1%, 13.0%, and 11.5% by ethidium bromide DNA by binding using albumin-containing, albumin-free, and Hank's solutions, respectively. Conclusions: Rat GMC with high and long viability can be isolated by low concentrations of pronase and EGTA. The adapted biochemical and microscopic methods are suitable for parallel examination of chemical injury of plasma membrane, mitochondria, and nuclei. Albumin increased ethanol 50% effective concentration to GMC. A combination of biochemical and morphological methods with ethanol-induced injury to GMC is proposed for direct cytoprotection investigations.

Structural organization of the Initial Lymphatics in the Monkey Mesentery and Intestinal Wall as Revealed byan Enzyme-Histochemical Method.

The structure and distribution of the initial lymphatics in whole mount preparations of the mesentery and intestinal walls of the Japanese monkey (Macaca fuscata) were studied using an enzyme-histochemical method (Kato et al., 1991, 1993). The lymphatic walls, colored dark brown by their positive 5'-nucleotidase (5'-Nase) activity, were clearly distinguished from the blood vessels (especially capillaries and arterioles) which were colored blue due to their positive alkaline phosphatase activity. The specificity and localization of both enzyme reactions were confirmed by comparative histochemical studies of the same specimen under a light microscope and scanning or transmission electron microscopes. Application of this staining method takes advantages of the overview preparation of flat membranous organs. The mesenterial area was generally lobulated in distribution with collecting lymphatics and blood vessels. At about the center of each lobule enclosed by the vessels, 5'-Nase-positive initial lymphatics assumed tubulo-saccular shapes, branching antler-like figure to form dense networks in the main lymph vascular pathway. Their apical parts revealed marked knob-like blind endings demarcated by a thin endothelial wall. No direct interconnection was recognizable between the lymphatic space and the tissue interstitium as a prelymphatic fluid pathway. In such preparations, 5'-Nase-positive lymphatic islands could be found isolated from the lymphatic network.

Enzyme-Histochemical Study on the Fine Distribution of the Intramural Lymphatics at the Ileocecal Junction of the Monkey Intestine

The fine distribution of the intramural lymphatics at the ileocecal junction of the monkey intestine, especially in the lamina propria of the ileocecal valve, was examined by light and electron microscopy using enzyme-histochemical staining. The distinction between the lymphatics and the blood vessels was made by light microscopy on cold glycol methacrylate resin (JB-4) sections using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) double staining. The lymphatics were found to show strong 5'-Nase activity and to comprise irregularly shaped vessels or spaces. The central lymphatic vessels (central lacteals) in low villi were seen to lie deep within the ALPase-positive subepithelial capillary network. In the ileum side of the ileocecal junction, the 5'-Nase-positive lymphatics were seen both in the superficial layer and the deep layer of the lamina propria. On the contrary, in the cecum side the mucosal lymphatics were less numerous in the superficial layer and were distributed mainly in the deep layer near the lamina muscularis mucosae. These lymphatics ran through the lamina muscularis and merged into the lymphatic network in the submucosa. The submucosal lymphatics communicated with each other at the ileocecal junction and formed a well-developed network. Collecting lymphatics with valves were also seen near the tunica muscularis (sphincter muscle) in the deep submucosa. These lymphatics traversed the muscle layer and drained into the subserosal lymphatics.

Purification to homogeneity of rat liver dinucleoside tetraphosphatase by affinity elution with adenosine 5′-tetraphosphate

Starting rom a partially purified dinucleoside tetraphosphatase (Np 4 Nase; EC 3.6.1.17), we developed an affinity elution purification protocol involving the strong competitive inhibitor adenosine 5′-tetraphosphate. Np 4 Nase bound to Cibacron Blue F3G-A-Sepharose 4B or to Reactive Blue 2-Sepharose CL-6B was specifically eluted with 10 μM adenosine 5′-tetraphosphate and 5 mM MgCl 2, but not by either of them separately. The final Np 4 Nase preparation was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Coomassie blue or silver staining. The protein band showed an apparent 18 kDa molecular mass. The specific activity of the homogeneous Np 4 Nase was about 150 units/mg, meaning a 45 000-fold increase and a 10% recovery with respect to the crude extract. After preparative polyacrylamide gel electrophoresis, protein visualization with KCl, fragmentation of the gel lane, and extraction, all the renatured Np 4 Nase activity was found associated to the 18 kDa band. The renatured enzyme showed the same K m value for diadenosine 5′,5′″- P 1, P 4-tetraphosphate as the partially purified or the native homogeneous Np 4 Nase.