Biomedical subjects

T B Knudsen

Publications and source records attributed to T B Knudsen.

At least 37 records · Page 2Linked to original sources

PubMed1996

Developing allantois is a primary site of 2'-deoxycoformycin toxicity.

In this study, an assessment of normal mouse allantoic development and its sensitivity to 2'-(R)-deoxycoformycin (dCF; Pentostatin) exposure were examined. Both dissecting microscopy and scanning electron microscopy were used to describe the normal growth and morphogenesis of the mouse allantois over gestational days 7-10 as a preliminary step in evaluating potential abnormal allantoic ontogeny and its effect on umbilical cord and placental development. Two abnormal allantoic/umbilical cord phenotypes were observed subsequent to injecting pregnant mice with 5 mg dCF/kg, i.p., on gestational day 7 (GD 7) and evaluating litters on GD 10, 11, and 12. Abnormal phenotypes included: (1) an allantois which extended approximately halfway across the exocoelom but failed to establish a functional contact with the chorion; and (2) a phenotype characterized by reduced expansion of the allantois across the chorionic surface, a very thin umbilical cord, and aberrant vascularization throughout the structure. Both abnormal phenotypes exhibited either an agenesis or hypogenesis of the umbilical cord and chorioallantoic plate, respectively. Neither abnormal phenotype, however, exhibited errors in the directionality of allantoic growth toward the chorion nor in the formation of aberrant contacts between allantois and adjacent yolk sac or amnionic mesenchyme. Statistical interpretation of the experimental data strongly suggested that abnormalities in allantoic/umbilical cord development were directly associated with embryolethality as evidenced by a decline in the frequency of abnormal allantoic/umbilical cord phenotypes over GD 10-12 (73, 36, and 4%; respectively) and a concomitant increase in the frequency of implantation site resorptions over the same time period (7, 47, and 78%). These results strongly suggest that the developing allantois is very sensitive to the effects of dCF exposure, and that interference with its development leads to embryolethality by GD 12.

Allantois↗

PubMed1995

Mutagenesis of human adenosine deaminase to active forms that partially resist inhibition by pentostatin.

Human adenosine deaminase (ADA) cDNA was randomly mutagenized in vitro and bacterial transformants were selected for resistance to the potent enzyme inhibitor, pentostatin (dCF). Cells transformed with mutant plasmids dCF-R2 and dCF-R6 were able to grow in the presence of 10(-6) M dCF, whereas 10(-11) M dCF blocked growth of cells complemented with wild-type ADA. DNA sequence analysis revealed double G/A conversions mutating Lys11 and Gln255 in dCF-R2, and Gly 31 and Glu 99 in dCF-R6, to different amino acids. Located far from the enzyme active site, these substitutions did not greatly affect the Km or Ki of the enzyme but were predicted to destabilize interactions within the enzyme structure. Mutants such as these may be useful to analysis of the stereology of inhibitor binding to ADA and toxicity of dCF.

Adenosine Deaminase↗

PubMed1995

Bcl-2 relieves deoxyadenylate stress and suppresses apoptosis in pre-B leukemia cells.

The influence of bcl-2 activity on 2'-deoxyadenosine-induced apoptosis was investigated in 697 human pre-B leukemia cells stably transfected with expression plasmid pHeBo-BCL-2alpha (697/BCL2 cells). Apoptosis was induced by the 2'-deoxyadenosine analogue, 2-chloro-2'-deoxyadenosine (Cl-dA), with the concentration for apoptosis in one-half of the cells at 24 hours (LD(50)) being 10 microM for 697 cells and 120 microM for 697/Bcl 2 cells. There was a strong positive correlation between Cl-dATP levels and apoptotic index (coefficient of determination, r(2)=0.95; P=0.027). When 697 cell and 697/Bcl 2 cell lines were treated with 5 microM Cl-dA, Cl-dATP did not significantly accumulate in the latter. The Cl-dATP/dATP ratio was 0.03 in Cl-dA treated 697/Bcl 2 cells but nearly 6 in treated 697 cells. Bcl 2 overproduction also suppressed the accumulation of dAMP, dADP and dATP in cells exposed to 2'-deoxyadenosine in the presence of pentostatin to abrogate the pronounced inversion of ATP/dATP pools associated with 2'-deoxyadenosine exposure. These results suggest that one consequence of bcl-2 activity is suppression of 2'-deoxyadenosine phosphorylation and elevation in the apoptotic target cells. Relief from deoxyadenylate stress imbalances implies a novel upstream site of bcl-2 activity.

Journal Article↗

PubMed1995

Production and characterization of multiple antigenic peptide antibodies to the adenosine A2b receptor.

A polyclonal antibody to the human adenosine A2b receptor (A2bR) was produced by immunizing a chicken with a multiple antigenic peptide consisting of eight copies of a 16-amino acid peptide, corresponding to the presumed second extracellular loop of the A2bR, linked to a branched lysine core. Western blotting with affinity-purified antibody revealed the human A2bR to be a protein of approximately 50-55 kDa, found in a variety of tissues including thymus, colon, and small intestine. The antibody also recognized mouse and rat A2bRs and revealed heterogeneity in size, with a 35-kDa protein being detected in small intestine in addition to the larger 50-52-kDa species in thymus, colon, and placenta. The chicken anti-human A2bR peptide antibody recognized the receptor in both frozen and formalin-fixed tissue sections. In human colon, the A2bR was highly expressed in epithelial cells of the crypts. A2bR immunoreactivity was also apparent in syncytiotrophoblast cells of human placental villi and in the basal zone of murine chorioallantoic placenta. These cell type-specific patterns of expression are consistent with the hypothesized roles of the A2bR in mediating electrogenic Cl- secretion and the resulting secretory diarrhea caused by colonic crypt abscesses and in regulating morphogenesis of the placenta. Insight into the multiple physiological consequences of A2bR engagement will be forthcoming from an analysis of the cell type-specific expression of this receptor in additional tissues.

Amino Acid Sequence↗

PubMed1994

Activation of apoptosis in early mouse embryos by 2'-deoxyadenosine exposure.

Adenosine deaminase (ADA) catalyzes the irreversible hydrolytic deamination of adenosine and deoxyadenosine to nontoxic derivatives. The importance of this reaction in the female reproductive tract of mice is suggested by pronounced utero-placental expression of ADA, and by embryolethality of the potent ADA-inhibitor deoxycoformycin (dCF) on day 7-8 of gestation. The present study investigated the effects of dCF, adenosine, and deoxyadenosine on the mouse neurula. Morphological cell death was monitored by the acridine orange reaction (AOR), and biochemical cell death by internucleosomal DNA cleavage (IDC). A strong AOR appeared in day 7-8 embryos between 3 and 4.5 hr post-exposure to dCF in utero; there was no apparent effect on day 6 or day 9 embryos. Most embryonic tissues were responsive, although the heart and extraembryonic membranes were resistant. Up to 75% of the embryonic chromatin was degraded in a regular pattern in concert with the AOR. Immediate activation of "whole-body" apoptosis was reproduced in short-term whole embryo culture with 0.1 mM deoxyadenosine in the presence of 0.01 mM dCF. This was not activated by exposure to dCF alone nor to adenosine; however, high concentrations of adenosine completely blocked the response to deoxyadenosine, whereas niacinamide inhibited the AOR without changing IDC. The cytotoxic effect of deoxyadenosine was correlated with an expansion of embryonic dATP pools determined by high-performance liquid chromatography analysis. The results suggest that deoxyadenosine is the embryotoxic metabolite which accumulates in the antimesometrium of pregnant mice treated with dCF. Exposure to this metabolic toxin activates apoptosis in day 7-8 embryos through an adenosine-sensitive, NAD-dependent mechanism.

Adenosine↗

PubMed1993

Murine ecto-5'-nucleotidase (CD73): cDNA cloning and tissue distribution.

The murine cDNA, encoding the purine catabolic enzyme, ecto-5'-nucleotidase (NT), was cloned and the tissue-specific distribution of both the mRNA and enzyme activity was examined. Starting with kidney RNA and primers based on the known rat sequence, reverse transcriptase-polymerase chain reaction (RT-PCR) was utilized to obtain the complete sequence for the translated portion of the murine cDNA. Murine NT is 94% identical to human NT at the amino acid (aa) level and 86% identical at the nucleotide (nt) level. NT enzyme assays revealed greater than tenfold more NT activity in mature vs. immature murine T- and B-lymphocytes. A similar increase in NT activity was also found when the pre-B-cell line, 70Z/3, was induced to produce surface kappa light chains with lipopolysaccharide (LPS) and gamma-interferon (gamma-IFN). Thus, culture systems in which murine lymphocytes mature may be useful for examining the mechanisms of NT gene regulation, as well as the function of NT in the immune system. In tissues, enzyme activity varied over 30-fold, from the lowest levels in skeletal muscle, thymus and spleen to highest in placenta, kidney and forestomach. Levels of mRNA, as determined by RNase protection assay, showed increased NT expression in the early gestation site, as compared to non-pregnant uterus, and in day-19.5 placenta, as compared to day-13 chorioallantoic placenta. Messenger RNA levels were in general proportional to enzyme activity, except in the lung and glandular stomach where mRNA levels were higher than expected, based on enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗

PubMed1993

Occurrence of embryotoxicity in mouse embryos following in utero exposure to 2'-deoxycoformycin (pentostatin).

Previous investigations had shown that i.p. injection of 2'-deoxycoformycin (dCF; pentostatin; 5 mg/kg) on either E7 or E8 into pregnant mice results in a 61-81% resorption rate at E17. The incidence of visible gross malformations among the surviving conceptuses was exceptionally low (3%) at the time of necropsy on E17 and was unrelated to dCF dose (Knudsen et al., Teratology, 40:5-626, '89; Teratology, 45:91-103, '92). These findings demonstrated the embryotoxicity of dCF but provided no clues as to the site(s) of dCF action. To define the lesion site(s), we have now examined embryos at 72 h (E10), 96 h (E11), and 120 h (E12) following administration of a highly embryotoxic dose of 5 mg dCF/kg to dams on E7. Deoxycoformycin caused multiple abnormalities and growth retardation, and the temporal sequence between maximal abnormal embryo incidence and resorption frequency was established. The quantitative data show that the maximal occurrence of abnormal embryos on E10 (71%) was followed by a maximal resorption rate on E12 (78%). There was a strong correlation (r = -0.82; P < 0.05) between the rapid decline of percent abnormal embryos over E10-E12 and the simultaneous increase in resorption rate, with linear regression analysis showing nearly equal but opposite slopes (-31.2% vs. +35.8% per gestational day, respectively). This suggests that one or more of the abnormalities seen at E10 is associated with the death and resorption of the embryo at E12. The dCF treatment perturbed a wide spectrum of developmental events, including neural tube closure, craniofacial and limb development, turning of the embryo, and growth retardation. None of the individual abnormalities, however, can quantitatively account for the high percentage of dead and resorbed embryos. Therefore, the specific cause of dCF-induced embryolethality is not clear. There is evidence both for direct dCF toxicity at specific embryonic sites as well as for a generalized retardation in the rate of development.

Abnormalities, Drug-Induced↗

PubMed1992

Adenosine levels in the postimplantation mouse uterus: quantitation by HPLC-fluorometric detection and spatiotemporal regulation by 5'-nucleotidase and adenosine deaminase.

Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryo-decidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5'-nucleotidase (5'-NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno-derivatized and quantitated by high-performance liquid chromatography-fluorescence detection (0.03 pmol/mg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmol/mg protein) and day 5 (0.59 nmol/mg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmol/mg protein) and day 7 (0.10 nmol/mg protein). The periimplantation adenosine surge coincided with uterine expression of 5'-NT, an enzyme which catalyzes the irreversible dephosphorylation of 5'-AMP to adenosine. 5'-NT expression was shown by Northern blot analysis to peak in the embryo-decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5'-NT activity was localized to the primary decidual zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5'-NT appeared on giant trophoblast (days 7-13) and the metrial gland (days 11-13). Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S-adenosylhomocysteine hydrolase) were not detected in the embryo-decidual unit suggesting that the net flux of utero-placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation. The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6-11), secondary giant cells (days 7-13), and spongiotrophoblasts (days 8-13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5'-NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities.(ABSTRACT TRUNCATED AT 400 WORDS)

5'-Nucleotidase↗

PubMed1992

Effects of (R)-deoxycoformycin (pentostatin) on intrauterine nucleoside catabolism and embryo viability in the pregnant mouse.

The viability of early mouse embryos is acutely sensitive to (R)-deoxycoformycin (pentostatin), a tight-binding inhibitor of adenosine deaminase (ADA). Previous studies have shown that a single 5-mg/kg dose on day 7 (plug = day 0) of gestation fully inhibits uteroplacental ADA activity within 0.5 h; causes massive cell death in the neural plate and primary mesenchyme by 6 h, major craniofacial anomalies by day 10, and resorption by day 12 (Knudsen et al., '89; Airhart et al., '91). The present study has examined further the developmental toxicity and early effects of this inhibitor on ADA metabolism. (R)-Deoxycoformycin was administered to pregnant CD-1 (ICR) mice as a single intraperitoneal dose of 0.5-10 mg/kg total body weight on days 6-11 of gestation. The major adverse effect, early resorption, was dose dependent and specific to day 7-8 exposure. Treatment with 5 mg/kg on day 7 resulted in 85% resorptions, 15% malformations, and a 24% reduction in mean fetal weight, whereas the same dose of (S)-deoxycoformycin had no effect. Levels of adenosine and 2'-deoxyadenosine, which are the endogenous substrates of ADA, were monitored in the embryo/decidual unit (E/D) by reversed-phase high-performance liquid chromatography (RP-HPLC). In response to the inhibitor, both nucleosides increased transiently in the antimesometrial compartment (antimesometrial decidua + embryo). Peak levels (Cmax) of adenosine and 2'-deoxyadenosine were dose dependent over the range tested (0.05-10 mg/kg). Exposure to 5 mg/kg on day 7 raised adenosine levels within 0.5 h to 42-fold over the basal level of 0.06 nmol/mg protein. There was an even stronger effect on 2'-deoxyadenosine levels, which were elevated 674-fold over the detection limit of 0.0005 nmol/mg protein. Direct exposure to the inhibitor in serum-free E/D culture produced similar results: 50 microM (R)-deoxycoformycin within 1 h raised adenosine levels 26-fold and 2'-deoxyadenosine levels 410-fold. In vivo studies also showed a general correlation between embryolethality and the length of adenine nucleoside pool expansion, apparent for exposure on day 7, 8, or 9 but not on day 6, suggesting that the embryo becomes sensitive to adenosine or 2'-deoxyadenosine once the neural plate has formed.

Adenosine↗

PubMed1992

Morphological analysis of abnormal digital chondrogenesis in the brachypod (bpH) mouse limb in organ culture.

Brachypod (bpH/bpH), an autosomal mutation in mice, is characterized by a shortening of the long bones and paws, and a delay or absence of ossification in some of the distal limb elements. The present study represents a detailed description of the brachypod phenotype in day 12 hindlimb buds maintained for 6 days in a submerged, serum-free organ culture system. Using this in vitro system, the proximal-to-distal effect on the severity of cartilage reduction was intensified in the brachypod explants with an intermediate expression in the heterozygotes. Immunofluorescent staining of the brachypod cartilage revealed a deficiency in and an abnormal distribution of the proteoglycans. Although there was no recognizable difference in the immunofluorescent staining for type II collagen between the mutant and wild-type, electron micrographs showed the presence of thick fibrils in the matrix. Other atypical structures in the brachypod cartilage included pleomorphic nuclei, reduced intracellular glycogen granules and profuse intercellular contacts. It is proposed that with the use of this in vitro system which supports the autonomous development of the individual limb elements, experiments concerning the pathogenesis of skeletal mutations such as brachypod should be more feasible.

Animals↗

PubMed1991

Ontogeny of adenosine deaminase in the mouse decidua and placenta: immunolocalization and embryo transfer studies.

This study has determined the cellular site of adenosine deaminase (ADA) expression in the mouse during development from Days 5 through 13 (day vaginal plug was found = Day 0) of gestation. Developmental expression of ADA progressed in two overlapping phases defined genetically (maternal vs. embryonal) and according to region (decidual vs. placental). In the first phase, ADA enzyme activity increased almost 200-fold in the antimesometrial region (decidua capsularis + giant trophoblast cells) from Days 6 through 9 of gestation but remained low in the mesometrial region. Immunohistochemical staining revealed a major localization of ADA to the secondary decidua. In the second phase, ADA activity increased several-fold in the placenta (labyrinth + basal zones) from Days 9 through 13 of gestation but remained low in the embryo proper. Immunohistochemical staining revealed a major localization of ADA to secondary giant cells, spongiotrophoblast, and labyrinthine trophoblast. Regression of decidua capsularis and growth of the spongiotrophoblast population accounted for an antimesometrial to placental shift in both ADA enzyme activity and a 40-kDa immunoreactive protein band. To verify a shift from maternal to fetal expression, studies were performed with two strains of mice (ICR, Eday) homozygous for a different ADA isozyme (ADA-A, ADA-B). Blastocysts homozygous for Adab were transferred to the uterus of pseudopregnant female recipients homozygous for Adaa. The isozymic pattern in chimeric embryo-decidual units analyzed at Days 7, 9, 11, and 13 revealed a predominance of maternal-encoded enzyme at Days 7 through 11 of gestation and a shift to fetal-encoded enzyme by Day 13. Thus, maternal expression of ADA in the antimesometrial decidua may play a role during establishment of the embryo in the uterine environment, whereas fetal expression of ADA in the trophoblast might be important to placentation.

Adenosine Deaminase↗

PubMed1991

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.

Adenosine Deaminase↗

PubMed1990

Axonal guidance of adenosine deaminase immunoreactive primary afferent fibers in developing mouse spinal cord.

This study examined the precision of central fiber growth in a subpopulation of dorsal root ganglion neurons in developing mouse spinal cord. Immunohistochemical techniques using a monospecific, polyclonal antiserum to mouse adenosine deaminase (ADA) were utilized to label a population of primary sensory afferents that have been found to exclusively innervate laminae I and II of the dorsal horn in adult mice. Initial growth of ADA-immunoreactive (ADA-IR) primary afferents occurred very early in development, embryonic day 10 (E10), a time coincident with the earliest settling time of dorsal root ganglion neurons. Adenosine deaminase immunoreactive primary afferents were observed throughout the cross-sectional area of the primordial dorsal funiculus (DF) as early as E10. Immunostained fibers remained quiescent in the DF during its growth and separation into the tract of Lissauer and dorsal column pathway. By E15, the two pathways had formed and ADA-IR fibers were observed exclusively in the tract of Lissauer. This segregation of fibers remained throughout development and reflected the adult pattern. Growth was reinitiated at E16 when the fibers advanced into the dorsal horn and proceeded directly to laminae I and II mimicking their adult distribution. Exuberant fiber growth was not detected throughout their development. These results strongly suggest that ADA-IR fibers exhibit precise fiber guidance to a preferred pathway, the tract of Lissauer, and accurate laminar innervation of the dorsal horn.

Adenosine Deaminase↗

PubMed1990

Developmental expression of adenosine deaminase in the upper alimentary tract of mice.

The distribution and localization of adenosine deaminase (ADA) was studied during postnatal development of the alimentary tract in mice. There was detectable enzyme activity in all organs examined, but a range of more than 10,000 fold in the relative levels of specific activity was observed among adult tissues. A comprehensive survey of multiple adult tissues revealed that the highest levels of ADA occur in the upper alimentary tract (tongue, esophagus, forestomach, proximal small intestine). Immunohistochemical analysis revealed that ADA was predominantly localized to the epithelial lining of the alimentary mucosa: the keratinized squamous epithelium that lines the forestomach, esophagus, and surface of the tongue; and the simple columnar epithelium of the proximal small intestine (duodenum, proximal jejunum). Biochemical analysis revealed that ADA was one of the most abundant proteins of these mucosal tissue layers, accounting for 5%-20% of the total soluble protein. Tissue-specific differences in ADA activity correlated both with levels of immunoreactive protein and RNA abundance. The level of ADA activity in the upper alimentary tissues was subject to pronounced developmental control, being low at birth and achieving very high levels within the first few weeks of postnatal life. The appearance in development of ADA-immunoreactivity coincided with maturation of the mucosal epithelium. These results suggest that ADA is subject to strong cell-specific developmental regulation during functional differentiation of certain foregut derivatives in mice.

Adenosine Deaminase↗

PubMed1989

Early postimplantation embryolethality in mice following in utero inhibition of adenosine deaminase with 2'-deoxycoformycin.

Adenosine deaminase (ADA) catalyzes the hydrolytic deamination of adenosine (or 2'-deoxyadenosine) to inosine (or 2'-deoxyinosine). Previously, we have shown that ADA activity is subject to strong cell-specific developmental regulation in placental tissues of mice between days 6 and 11 of gestation (Knudsen et al.:Biology of Reproduction 39:937-951, 1988). In the present study, we examined the effects of intrauterine exposure to 2'-deoxycoformycin (dCF; pentostatin), a potent irreversible inhibitor of ADA, on early postimplantation development. Deoxycoformycin was administered to pregnant ICR mice as a single intraperitoneal injection at a dose of 5 mg/kg on one of days 6 through 11 of gestation (plug day 0). A marked increase in the incidence of implantation site resorptions was observed following treatment specifically on days 7 (61% resorbed) or 8 (78% resorbed). No effect was observed following treatment on days 6, 9, 10, or 11. ADA-immunoreactive protein was shown, by ABC-immunoperoxidase staining on days 7 or 8 of gestation, to be present at high levels in decidual cells of the antimesometrial region but at below-detectable levels in the embryo. Treatment of pregnant dams with dCF on day 7 produced a complete (greater than 99%) inhibition of ADA activity in the antimesometrial decidua by 30 min, induced excessive cell death in the prospective neural plate and primary mesenchyme of the trilaminar disc by 6 h, and arrested embryonic development at an early somite stage. These results suggest that the antimesometrial decidua plays a protective role in preventing an inappropriate accumulation of endogenous ADA substrates in the implantation site.

Adenosine Deaminase↗

PubMed1988

Alterations in cell surface galactosyltransferase activity during limb chondrogenesis in brachypod mutant mouse embryos.

The autosomal mutation brachypod (bpH/bpH) in the mouse affects the development of precartilage mesenchymal condensation in the limb-bud. We have previously shown that this defect is localized to the expression of terminal N-acetylglucosamine (GlcNAc) glycoproteins in the plasma membrane (Elmer and Wright, '83). The present study is focused on cell surface galactosyltransferase (GalTase), an ectoenzyme that transfers galactose to its GlcNAc substrate. Purified plasma membrane preparations derived from wild-type (+/+), heterozygote (+/bpH) and brachypod (bpH/bpH) embryonic mouse limb cells were assayed for GalTase activity during in vitro and in utero chondrogenesis using High-Performance Liquid Chromatography (HPLC). On embryonic day E12, prior to overt expression of the mutant gene, no significant difference in GalTase activity was observed. By the third day in culture, all major chondrogenic elements of the autopod were present in +/+ and +/bpH embryos, whereas the mutant autopods were markedly deficient in staining and appeared consistently shorter. The accumulation of alcianophilic cartilage matrix in the wild-type was accompanied by a 29% increase in GalTase activity, which reflected the net change (29%) observed during development from days E12 to E13 in utero. The GalTase activity for the in utero E13 mutant (13%) was significantly different from control. In culture, day E12 mutant autopods actually decreased in their GalTase level by 3 days so that the activity was reduced to only 57% of the wild-type. Though GalTase activity in the heterozygote showed an intermediate expression, optical image analysis did not reveal consistent differences in cartilage development when compared to +/+, arguing against a gene-dosage effect at the gross anatomical level. These data indicate that an increase in plasma membrane GalTase activity is a natural developmental event that occurs during limb-bud chondrogenesis and a decrease in GalTase activity contributes to the dysmorphogenesis in brachypod limb-buds.

Animals↗

PubMed1988

Developmental expression of adenosine deaminase in placental tissues of the early postimplantation mouse embryo and uterine stroma.

In this study, we have investigated the distribution of adenosine deaminase (ADA) in embryonic, extra-embryonic, and decidual tissues of the developing mouse embryo. ADA catalyzes a key step in purine metabolism converting adenosine to inosine. ADA specific activity (nmol/min/micrograms protein) was present at low levels in the embryo-decidual unit during the first 2 days of postimplantation development but then increased starting late on Day 6 of gestation (Day 0 plug). By Day 9, ADA specific activity was 80-fold higher than on Day 6. A histochemical staining method for ADA activity was applied to cryostat sections of the implantation site. The developmental increase localized primarily to the trophoblast/antimesometrial decidua interface between Days 7 and 9 of gestation, and decidua basalis and the metrial gland by Day 11. Immunofluorescent staining with sheep anti-mouse ADA antiserum confirmed the presence of ADA antigenicity in tissues forming the maternal/fetal interface. ADA specific activity was 19-fold higher in homogenates of the Day 11 decidua/parietal yolk sac than in the thymus, a tissue generally thought of as ADA-rich. High levels of ADA activity and immunoreactivity were also detected in the embryonal plasma during organogenesis, but the embryo proper showed only low levels. These results indicate that ADA is tightly regulated within tissues forming the maternal/fetal interface during early postimplantation stages of development.

Adenosine Deaminase↗

PubMed1987

Evidence for negative control of growth by adenosine in the mammalian embryo: induction of Hmx/+ mutant limb outgrowth by adenosine deaminase.

We investigated the growth-regulatory actions of adenosine and adenosine deaminase (ADA) during embryonic limb development in the mouse. Polydactylous outgrowth, an expression of the Hemimelia-extra toe (Hmx/+) mutant phenotype, was experimentally regulated in hindlimb buds explanted into a serum-free in vitro system at stage 18 of gestation. Its expression was promoted by exposure to 0.1 or 0.2 IU/ml exogenous ADA and suppressed by co-exposure to 10 nM (-)-N6-(R-phenylisopropyl)-adenosine (N6-PIA). Evidence that N6-PIA acted as a high-affinity agonist against the external adenosine receptor was provided by experiments in which 100 microM caffeine, a known antagonist, competitively blocked its effect. The endogenous adenosine content was analyzed by reversed-phase high-performance liquid chromatography with fluorometric detection following its conversion to the 1,N6-ethenoadenosine derivative. At stage 18, the adenosine levels were 0.5 pmol/micrograms DNA in whole embryos and 0.08 pmol/micrograms DNA in hindlimb buds. At the same stage, partially purified extracts of the embryonal plasma enriched fraction contained high levels of ADA activity (0.04-0.06 IU/embryo, or 0.7-1.0 IU/mg protein). In contrast, blood cells contained 0.0001 IU/embryo (or 0.01 IU/mg protein). This enzyme occurred as a single kinetic form with a molecular weight of 45000-47000 daltons and an apparent Km of 36-38 microM. Its presence in the embryonal plasma argues against an endocrine mechanism of adenosine secretion in favor of autocrine (self-regulatory) or paracrine (proximate-regulatory) mechanisms. Taken together, our results suggest that the in vitro outgrowth of the prospective polydactylous region is induced upon escape from the local growth-inhibitory influence of extracellular adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗