Developmental expression of adenosine deaminase during decidualization in the rat uterus

Adenosine deaminase (ADA) is expressed in high concentrations at the fetal-maternal interface during postimplantation stages of gestation in the mouse. The experiments reported here were designed to identify the specific uterine cells that express ADA subsequent to implantation in the rat and to determine if embryonic cells contribute to ADA expression. The results of biochemical analysis demonstrate that ADA-specific activity increases to very high levels in implantation sites, beginning approximately 72 h after blastocyst attachment. Immunocytochemical analysis localized this ADA expression to the decidualized stromal cells in the antimesometrial region of the pregnant uterus. In experimentally induced deciduoma, these cells were capable of synthesizing high levels of both ADA and mRNA for ADA in the absence of embryos. The enzyme first appeared in decidual cell cytoplasm, approximately 72 h after induction of decidualization, and later was localized in the decidual cell nuclei. Since the expression of ADA and its mRNA in decidual cells follows the appearance of desmin, a protein marker for decidualization, by at least 48 h, ADA appears to be involved in the functioning of mature decidual cells rather than in stromal cell differentiation. The expression of ADA, but not desmin, was restricted to the antimesometrial decidual cells and decreased when these cells regressed. At mid-gestation ADA activity increased and was localized principally in the fetal placenta. The results presented here demonstrate that ADA is localized to the antimesometrial decidual cell and that its expression is consequent to differentiation of the uterine stromal cell and independent of any embryonic stimulus.     

Immunomorphological analysis of G2-chalone localization during the process of rat epidermis histogenesis

The appearance of G2-chalone in the cytoplasm of the intermediate cell layer and partly in the periderm of 17-day-old rat embryo epidermis has been demonstrated by the indirect method of Coons using a monospecific antiserum. G2-chalone was absent from the basal cell layer of 17--21-day-old embryos and of the newborn rats. It was found in all the epidermal layers in 2--5-day-old postnatal rats, while in 6--9-day-old animals it was primarily detected in the cytoplasm of spinous and basal cells. Thus the localization of epidermal G2-chalone typical for defined tissue becomes stabilized at the end of epidermis histogenesis.     

Distribution of adenosine deaminase in some rat tissues. Inhibition by ethanol and dimethyl sulfoxide

The level of adenosine deaminase in various rat tissues has been tested. The enzyme activity of cytosolic fractions decreased in the following order: lung greater than spleen greater than small intestine greater than stomach greater than kidney greater than heart greater than liver greater than skeletal muscle greater than forebrain greater than cerebellum. The enzyme had identical patterns from tissue to tissue with respect to Km, V, and Ki values for ethanol and for dimethyl sulfoxide, with respect to electrophoretic behaviour and to inhibition by antibodies anti-rat brain adenosine deaminase.     

Distribution of adenosine deaminase complexing protein (ADCP) in human tissues

The normal distribution of adenosine deaminase complexing protein (ADCP) in the human body was investigated quantitatively by ADCP-specific radioimmunoassay (RIA) and qualitatively by immunohistochemistry. In these studies we used a specific rabbit anti-human ADCP antiserum. In all 19 investigated tissues, except erythrocytes, ADCP was found by RIA in the soluble and membrane fractions. From all tissues the membrane fractions contained more ADCP (expressed per mg protein) than the soluble fractions. High membrane ADCP concentrations were found in skin, renal cortex, gastrointestinal tract, and prostate. Immunoperoxidase staining confirmed the predominant membrane-associated localization of the protein. In serous sweat glands, convoluted tubules of renal cortex, bile canaliculi, gastrointestinal tract, lung, pancreas, prostate gland, salivary gland, gallbladder, mammary gland, and uterus, ADCP immunoreactivity was found confined to the luminal membranes of the epithelial cells. These data demonstrate that ADCP is present predominantly in exocrine glands and absorptive epithelia. The localization of ADCP at the secretory or absorptive apex of the cells suggests that the function of ADCP is related to the secretory and/or absorptive process.     

Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit brain

Adenosine deaminase and adenosine deaminase complexing protein have been localized in rabbit brain. Brains fixed in paraformaldehyde or in Clarke's solution were blocked coronally. Blocks from brains fixed in paraformaldehyde were either frozen in liquid nitrogen or embedded in paraffin. Tissue fixed in Clarke's solution was embedded in paraffin. Sections from each block were stained by the peroxidase-antiperoxidase method for adenosine deaminase or complexing protein using affinity-purified goat antibodies. Adenosine deaminase and complexing protein did not co-localize. Adenosine deaminase was detected in oligodendroglia and in endothelial cells lining blood vessels, whereas complexing protein was concentrated in neurons. The subcellular location and appearance of the peroxidase reaction product associated with individual cells was also quite distinctive. The cell bodies of adenosine deaminase-positive oligodendroglia were filled with intense deposits of peroxidase reaction product. In contrast to oligodendroglia, the reaction product associated with most neurons stained for complexing protein was concentrated in granular-appearing cytoplasmic deposits. In some instances, these deposits were clustered about the nuclear membrane. Staining of neurons in the granular layer of cerebellum was an exception. Granule cells were lightly outlined by peroxidase reaction product. Cerebellar islands, also referred to as glomeruli, were stained an intense uniform brown. These results raise the possibility that oligodendroglia and blood vessel endothelia, through the action of adenosine deaminase, might play a role in controlling the concentration of extracellular adenosine in brain. They do not, however, support the suggestion that complexing protein aids in adenosine metabolism by positioning adenosine deaminase on the plasma membrane.     

Conformational change of adenosine deaminase during ligand-exchange in a crystal

Adenosine deaminase (ADA) perpetuates chronic inflammation by degrading extracellular adenosine which is toxic for lymphocytes. ADA has two distinct conformations: open form and closed form. From the crystal structures with various ligands, the non-nucleoside type inhibitors bind to the active site occupying the critical water-binding-position and sustain the open form of apo-ADA. In contrast, substrate mimics do not occupy the critical position, and induce the large conformational change to the closed form. However, it is difficult to predict the binding of (+)-erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as it possesses characteristic parts of both the substrate and the non-nucleoside inhibitors. The crystal structure shows that EHNA binds to the open form through a novel recognition of the adenine base accompanying conformational change from the closed form of the PR-ADA complex in crystalline state.     

Amplification of adenosine deaminase gene sequences in deoxycoformycin-resistant rat hepatoma cells

Deoxycoformycin-resistant rat hepatoma cells exhibit up to a 2000-fold increase in adenosine deaminase activity compared to the sensitive parental cells. The increased enzyme activity in these cells is accompanied by similar increases in 1) the amount of adenosine deaminase protein, 2) the relative rate of adenosine deaminase synthesis in vivo, and 3) adenosine deaminase mRNA activity. To further investigate the mechanism(s) responsible for the overproduction of adenosine deaminase in these cells, we have isolated a recombinant plasmid containing a 1.4-kilobase insert complementary to at least part of the adenosine deaminase mRNA. Using this cDNA as a specific hybridization probe, all deoxycoformycin-resistant variants were shown to have increased amounts of adenosine deaminase mRNA and gene sequences. The relative increase in the level of mRNA and gene copy number was similar to the relative increase in enzyme activity for most resistant cell lines. However, the degree of adenosine deaminase gene amplification in one deoxycoformycin-resistant cell line (6-10-200) was 3-4-fold less than the relative increase in adenosine deaminase mRNA. These results indicate that the increased adenosine deaminase activity in deoxycoformycin-resistant rat hepatoma cells is due in large part, but not exclusively, to gene amplification.     

The effect of benzene on the activity of adenosine deaminase in tissues of rats

Swiss Albino (Rat rattus norvegicus) rats were intraperitoneally injected with a 100 mg kg(-1) dosage of benzene, a toxic and carcinogenic agent widely used for industrial purposes. Changes in the adenosine deaminase (ADA) activity in the liver, kidney and serum of rats were investigated at 0, 2, 4, 8, 16, 32 and 64 h following injection. Serum physiological was administered to each control group. Enzyme activities were measured spectrophotometrically. Our purpose was to further investigations of some diseases caused by benzene, and present evidence of variations in the activity of ADA enzyme effected by benzene. While benzene caused significant inhibitions in ADA activity in the liver at 16 and 32 h and at 0.05 probability level, no significant inhibition or activation occurred at other test periods (hours). ADA activity did not present any significant variation in the kidneys. It was observed that ADA activity displayed similar patterns in the control groups. Comparisons of ADA activities in the two groups showed a statistically significant decrease between 4(th) and 64(th) hours (p< 0.05), demonstrating a direct correlation between benzene and its effects on ADA enzymes.     

Olfactory marker protein during ontogeny: Immunohistochemical localization

Specific immunohistochemical staining for the olfactory marker protein (OMP) is first demonstrated in rat olfactory receptor neurons at embryonic day 18, at which age no OMP can be seen in the olfactory bulb or vomeronasal epithelium. At 21 days OMP staining in the olfactory epithelium is more extensive and is evident in the fibrous and glomerular layers of the bulb as well. Staining intensity increases progressively until the full adult pattern is seen by 1 month postnatally. In the vomeronasal organ, staining is not observed until the fourth postnatal day and, even then, only with higher antiserum concentrations. In mice, very similar results are obtained, except for a much earlier appearance of OMP, on embryonic day 14. Olfactory epithelium from 12- and 13-day rat embryos maintained in organ culture for up to 2 weeks did not exhibit OMP staining, nor did several neural or nonneural tissues from adult animals. The temporal and causal interrelationships between OMP and other indicators of olfactory receptor cell maturation are considered.     

Developmental changes of adenosine deaminase, xanthine oxidase, and uricase in mouse tissues

The activities of adenosine deaminase, xanthine oxidase, and uricase were followed in the liver, kidney, stomach, and intestine during pre- and postnatal development of the mouse. Results indicated that some type of coordinate control exists between the uricase and the xanthine oxidase levels in liver, stomach, intestine, and kidney. No coordinate control was seen between adenosine deaminase and xanthine oxidase in liver and kidney. The developmental changes between the intestine and stomach xanthine oxidase and adenosine deaminase were found to be related. The results obtained were consistent with the idea that intermediate metabolites in a pathway play some role in controlling the level of enzymes further down the pathway.A superificial resemblance in the timing of changes in feeding habits and changes in enzyme levels during development was found. Results of artifical change of feeding habit indicate that the control of enzyme levels was inherent rather than the result of dietary change.     

Effect of benzodiazepines on the activity of AMP deaminase and adenosine deaminase in rat brain tissue in vivo

Phenazepam (5 mg/200 g) and seduxen (3 mg/200 g) injected intraperitoneally to 184 rats altered AMP-deaminase and adenosine deaminase brain activity. Seduxen was observed to increase AMP-deaminase and adenosine deaminase activity by 89.1% and 32.4%, respectively an hour after the injection. Phenazepam increased the activity of the enzymes by 35.5% and 38.5%, respectively two hours after the injection. The effect is suggested to be due to de novo benzodiazepine-induced enzyme synthesis.     

The distribution of adenosine deaminase among lymphocyte populations in the rat.

Adenosine deaminase (ADA) activity was determined in young rat lymphocyte populations. The ADA-specific activity (per 10(8) cells and per milligram protein) was 3- to 10-fold higher in thymocytes than in lymphocytes from thoracic duct, lymph node, spleen, and bone marrow. The high ADA activity in thymocytes appeared to be preferentially associated with cortical thymocytes. Enrichment or depletion of cortical thymocytes by density gradient centrifugation, cortisone treatment, or selective lysis with anti-Thy-1 plus complement resulted in parallel increases or decreases in ADA levles. These results also suggested that medullary thymocytes have ADA levels similar to those of peripheral lymphocytes. "Immature" cortical thymocytes and thymocyte progenitors appeared to have low ADA activity; low enzyme levels were found in fetal thymus at 16 days of embryonic life, in the early phases of thymus regeneration, and in a "null" cell population isolated from bone marrow. This study demonstrates that ADA activity varies markedly during T lymphocyte differentiation and suggests that fundamental differences in nucleotide metabolism may exist in T cells at different stages of development.     

Effect of exercise on adenosine deaminase activity in rat skeletal muscles.

Adenosine deaminase activity was shown to decrease in each skeletal muscle type (the slow-twitch oxydative, fast-twitch oxydative--glycolytic and fast-twitch glycolytic) at the beginning of exercise of moderate intensity and to return to the control when exercise was continued till exhaustion. 5 min occlusion of the femoral artery had no effect on the enzyme activity in either muscle. The reduction of the enzyme activity at the onset of exercise could result in reduction of adenosine breakdown and thus contribute to vasodilation at this stage of increased contractile activity of the muscles.     

Immunohistochemical localization of metallothionein in developing rat tissues

Metallothionein (MT) is a cysteine-rich, low molecular weight protein inducible by heavy metal ions and various endogenous factors. Using an indirect immunofluorescent technique, we studied the localization of MT in developing rat tissues (kidney, small intestine, and liver). In kidney of the neonate and fetus, MT was found in both the cytoplasm and the nucleus of renal tubular epithelia. Localization of MT changed with shift of zonation in the renal cortex during development. Metallothionein was found mainly in the inner zone of the cortex but not in tubules of the neogenic zone on Day 4. Until Day 18, tubular cells containing MT were observed in a part of the cortex adjacent to the medulla, followed by a significant decrease in immunostaining by Day 27. In small intestine of the neonate, MT was localized predominantly in Paneth and goblet cells which play secretory roles. The number of goblet cells with strong immunostaining for MT was maximal on Day 27. In liver of 20-day fetuses and of 4-day-old neonates, both the cytoplasm and the nucleus of hepatocytes exhibited strong immunofluorescence. The intensity of MT staining diminished with development, and by 18-27 days after birth no immunofluorescence was observed in the nucleus. We further studied a possible association of MT with development by localizing MT in livers obtained from partially hepatectomized and laparotomized rats. Hepatectomy led to the appearance of MT not only in the nucleus and cytoplasm of hepatocytes but also in sinusoids and bile canaliculi. After laparotomy, MT immunofluorescence was observed only in the cytoplasm. The present results suggest a possible involvement of MT in cell proliferation and differentiation, as well as in transport and secretion of this metal-binding protein.