Mammalian Brain Alkaline Phosphatase: Expression of Liver/Bone/Kidney Locus Comparison of Fetal and Adult Activities

The alkaline phosphatases (EC 3.1.3.1) are determined by at least three gene loci, which can be sharply distinguished one from another by their sensitivity to inhibition with various amino acids and peptides and by ther-mostability. Alkaline phosphatase is present in the brains of guinea pig, rat, mouse, hamster, squirrel, rabbit, cat, sheep, cow, tamarin, baboon, and man. The gene locus coding for alkaline phosphatase in all these brains is the liver/ bone/kidney locus, as indicated by thermostability studies and by inhibition studies with L-phenylalanine, L-homoarginine, and L-phenylalanylglycylglycine. The average brain alkaline phosphatase activity is about 35% of the average for the livers and only 7.2% and 4.4% of the average kidney and placental activities, respectively. During growth and development, brain alkaline phosphatase activity decreases in the mammals studied. The amount of change is tissue- and species-dependent.     

Studies on conversion of labelled pregnenolone to progesterone by olive baboon placental cytotrophoblast cells in vitro

The experiments were designed to study the influence of a variety of substances, which have been reported to affect placental steroid metabolism, on pregnenolone metabolism by olive baboon placenta cells. Placentae were obtained from six baboons by caesarian section between 100 and 110 days of pregnancy. The cells were isolated by enzyme digestion and Ficoll gradient separation and incubated in Ham's F10 media with 5μ Ci [4,7, — 3H] pregnenolone in the presence or absence of indomethacin (0.1 mmol/1), dibutyryl cAMP (10 mmol/l), phorbol myristic acetate (10 nmol/l), 3-isobutyl-1-methylaxanthine (0.5 mmol/l), calcium ionophore A23187 (1 μmol/l), and nordihydroguaiaretic acid (40 μg/ml). Control experiments were done using leucocytes, inactivated placental cells and Ham's F10 media. Time course and dose response studies were also done. Placental cells converted pregnenolone to progesterone in a dose related manner. Addition of test compounds did not affect conversion rates. It is concluded that further studies are needed for elucidation of mechanisms that regulate progesterone synthesis in baboon placentae.     

Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.     

Lingual and gastric lipases: species differences in the origin of prepancreatic digestive lipases and in the localization of gastric lipase

The source of the lipase(s) acting in the stomach was investigated in five animal species: rat, mouse (rodents), rabbit (lagomorphs), guinea pig (caviidae), baboon and human (primates). The activity of lingual and gastric lipases was quantitated in homogenates of lingual serous glands and of gastric mucosa, respectively, by the hydrolysis of tri[3H]oleylglycerol and is expressed in units/g (1 U = 1 mumol [3H]oleic acid released/min) per g tissue wet weight, mean +/- S.E. There were marked differences in the activity level of lingual and gastric lipases among species: mouse and rat had high levels of lingual lipase activity (250 +/- 20 and 824 +/- 224 U/g) and only traces of gastric lipase activity (4.5 +/- 0.9 and 0.04 U/g, respectively), whereas rabbit and guinea pig had no lingual lipase activity and only gastric lipase activity (78 +/- 48 and 27 +/- 7.4 U/g, respectively). In the baboon and human, gastric lipase was the predominant enzyme (109 +/- 20 U/g and 118 +/- 8.8 U/g, respectively), whereas lingual lipase activity was present in trace amounts only (0.04 U/g and 0.3 U/g, respectively). In addition to species differences in the origin of the preduodenal lipases, there were also species differences in the distribution of gastric lipase in the stomach. Thus, while in the rabbit, gastric lipase was localized exclusively in the cardia and body of the stomach, it was diffusely distributed in the entire stomach of the guinea pig and baboon. A comparison between the level of activity of lipase and pepsin (the two chief digestive enzymes secreted by the stomach), showed differences in their localization in the species studied. The difference in source (tongue vs. stomach) and site (cardia-body vs. entire stomach) of lipase secretion must be taken into account in future studies of these digestive enzymes. Although the exact contribution of lingual and gastric lipases individually to fat digestion in species which contain both enzymes cannot yet be evaluated, the markedly higher levels of gastric lipase activity in the baboon and human suggests that, in primates, gastric lipase is probably the major non-pancreatic digestive lipase.     

Protein binding of cortisol by means of competitive adsorption: application to cortisol binding by serum of sixteen eutherian mammals.

1. Binding of 3H-cortisol by serum proteins by means of competitive adsorption was relatively high by serum of the gerbil, human, rabbit, sheep, tree shrew, hamster, rhesus monkey and horse. 2. A somewhat lower binding was observed by serum proteins of the baboon, cattle, dog, rat and cat. 3. Serum taken from either the mouse, guinea pig or pig gave very flat binding curves, specific binding not exceeding 5% of added 3H-cortisol. 4. It is concluded that the measurement of protein-binding of 3H-cortisol by means of competitive adsorption is a reliable method for serum of most eutherian species but is unsuited if serum of the mouse, guinea pig or pig is used.     

Effects of retionoids on invasion of organ cultures of chick chorioallantoic membrane by adenovirus transformed cells

Summary Invasion of chick chorioallantoic membrane (CAM) organ cultures by rat 3Y1 cells transformed by the highly oncogenic human adenovirus type 12 (3Y1/12-10 cells) was inhibited by several retinoids tested. The anti-invasive activity of the retinoids was dependent on retinoid concentration and continuous (4d) exposure of the CAM. The 50% retinoid dose (dose effective in achieving a response in half of the organ cultures) that inhibited invasion was 0.85 μg/ml of retinol palmitate, 0.39 μg/ml of retinoic acid, or 0.16 μg/ml of retinol acetate. This dose was of the same order of magnitude as that which induced CAM differentiation, and was three-to fourfold less than the dose that caused cytotoxic damage of CAM. In addition, the retinoids inhibited 3Y1/12-10 cell growth by approximately 40% at levels over 10-fold higher than those needed for anti-invasion activity. The findings suggest that the anti-invasive activity of retinoids was at least partly due to direct induction of cell differentiation of the CAM host tissue. This work was supported by National Cancer Institute Grant CA 13231 and by University of Akron Grant RG 832.     

Species variation in lung antioxidant enzyme activities

Exposure of several different animal models to O2-induced lung injury has revealed marked differences in sensitivity of various species to O2 damage. These differences may be due in part to variation of cellular antioxidant defenses. To characterize lung antioxidant enzyme activities in different species, we measured lung activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GSH S-trans) in rat, hamster, baboon, and human lung. Soluble lung fractions were also fractionated on Sephadex G-150-S columns and GSH-Px activity was measured using both cumene hydroperoxide and H2O2. This was done to evaluate non-Se-dependent GSH-Px activity in these lung samples. Human lung was obtained at surgery from patients undergoing lobectomy or pneumonectomy for localized lung tumors. SOD activity was similar for all four groups. GSH-Px activity was higher in rat lung than baboon or hamster lung. Lung CAT activity was variable with the highest activity present in the baboon which revealed a lung CAT activity 10 times higher than activity present in the rat. Lung GSH S-trans activities were higher in hamster, baboon, and human lung than in rat lung. Non-Se-dependent GSH-Px was present in rat lung but absent in hamster, baboon, and human lung. We conclude that the hamster was the best model of the animals studied for mimicking human lung antioxidant enzyme activities. Rat lung antioxidant enzyme activities were markedly different from any of the other species examined.     

Forskolin dilates the maternal ovine placental vascular bed

We have examined the placental vascular responses to forskolin in 8 near-term sheep. The drug was administered for 5 min at 1 ml/min of 10(-3) M forskolin via a retrograde uterine arterial catheter. Blood flows were measured with radioactive microspheres. Forskolin increased the nonplacental uterine blood flow from 0.318 +/- 0.031 (SEM) to 0.738 +/- 0.071 ml/min per g of tissue, P less than 0.001. The nonplacental uterine vascular resistance decreased from 308 +/- 26 to 132 +/- 12 mmHg/ml/min per g, P less than 0.001. Forskolin increased the placental blood flow from 1.8 +/- 0.18 to 2.08 +/- 0.16 ml/min per g of tissue, P less than 0.05. The placental vascular resistance decreased from 54.7 +/- 5.1 to 45.9 +/- 3.7 mmHg/ml/min per g, P less than 0.03. In the same animals we then infused angiotensin II at 5 micrograms/min via the jugular vein to induce placental vasoconstriction. In this series, the forskolin increased the nonplacental uterine blood flow from 0.141 +/- 0.016 to 0.485 +/- 0.079 ml/min per g of tissue, P less than 0.001, and the uterine vascular resistance decreased from 968 +/- 104 to 283 +/- 36 mmHg/ml/min per g, P less than 0.001. The placental blood flow increased from 2.08 +/- 0.012 to 2.69 +/- 0.17 ml/min per g of tissue, P less than 0.01 and placental vascular resistance decreased from 61.9 +/- 4.4 to 46.0 +/- 3.7 mmHg/ml/min per g, P less than 0.001.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development

Oviduct-specific glycoprotein (oviductin) plays an important role during fertilization and early embryonic development. The oviductin cDNA was successfully cloned and sequenced in goat, which possessed an open reading frame of 1620 nucleotides representing 539 amino acids. Predicted amino acid sequence showed very high identity with sheep (97%) followed by cow (94%), porcine (77%), hamster (69%), human (66%), rabbit (65%), mouse (64%) and baboon (62%). The bioinformatics analysis of the sequences revealed the presence of a signal sequence of 21 amino acids, one potential N-linked glycosylation site at position 402, 21 potential O-linked glycosylation sites and 36 potential phosphorylation sites. The native oviductin was purified from the oviductal tissue, which showed three distinct bands on SDS-PAGE and western blot (MW ∼60-95 kDa). The predicted molecular weight of goat oviductin was 57.5 kDa, calculated from the amino acid sequences. The observed higher molecular weight has been attributed to the presence of large number of potential O-linked glycosylation sites. The lower concentration (10 μg/mL) of oviductin increased the cleavage rate, morula and blastocyst yield significantly (P < 0.05) as compared to higher concentration (100 μg/mL). Goat oviductin retarded the activity of pronase (0.1%) on zona solubility of oocytes significantly (P < 0.01).     

Gene expression in placentation of farm animals: an overview of gene function during development

Eutherian mammals share a common ancestor that evolved into two main placental types, i.e., hemotrophic (e.g., human and mouse) and histiotrophic (e.g., farm animals), which differ in invasiveness. Pregnancies initiated with assisted reproductive techniques (ART) in farm animals are at increased risk of failure; these losses were associated with placental defects, perhaps due to altered gene expression. Developmentally regulated genes in the placenta seem highly phylogenetically conserved, whereas those expressed later in pregnancy are more species-specific. To elucidate differences between hemotrophic and epitheliochorial placentae, gene expression data were compiled from microarray studies of bovine placental tissues at various stages of pregnancy. Moreover, an in silico subtractive library was constructed based on homology of bovine genes to the database of zebrafish — a nonplacental vertebrate. In addition, the list of placental preferentially expressed genes for the human and mouse were collected using bioinformatics tools (Tissue-specific Gene Expression and Regulation [TiGER] — for humans, and tissue-specific genes database (TiSGeD) — for mice and humans). Humans, mice, and cattle shared 93 genes expressed in their placentae. Most of these were related to immune function (based on analysis of gene ontology). Cattle and women shared expression of 23 genes, mostly related to hormonal activity, whereas mice and women shared 16 genes (primarily sexual differentiation and glycoprotein biology). Because the number of genes expressed by the placentae of both cattle and mice were similar (based on cluster analysis), we concluded that both cattle and mice were suitable models to study the biology of the human placenta.     

Hamster D-amino-acid oxidase cDNA: rodents lack the 27th amino acid residue in D-amino-acid oxidase

Summary.  The nucleotide sequence of cDNA that encodes hamster d-amino-acid oxidase (DAO) was determined. The cDNA consisted of 1,590 nucleotides and a poly(A) tail. It had an open reading frame for a protein consisting of 346 amino acid residues. The number of the amino acid residues is the same as that of the rat DAO. However, the hamster DAO has one residue more than mouse DAO and one residue less than human, pig, rabbit, and guinea pig DAOs. Amino acid sequence of the hamster DAO was highly similar to those of mouse and rat DAOs: 89% and 88% of the amino acid residues were identical between the hamster and mouse DAOs and between the hamster and rat DAOs, respectively. The homology was slightly less between the hamster DAO and the human (81%), pig (78%), rabbit (78%), or guinea pig DAO (82%). It has been proposed that the mouse and rat DAOs lack an amino acid residue corresponding to the 25th residue of the DAOs of other mammals. However, a detailed comparison of the amino acid sequences as well as the underlying nucleotide sequences by inclusion of the hamster ones revealed that the rodent DAOs does not lack the 25th, but the 27th residue. Received January 16, 2002 Accepted June 20, 2002 Published online November 14, 2002 Authors' address: Dr. Ryuichi Konno, Department of Microbiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan, Fax: +81-282-86-5616     

Bovine placentomes contain factors which decrease progesterone secretion

We examined the effects of 20% ammonium sulfate precipitates from cytosolic extracts of whole placental tissue collected between 100-150 days of gestation on progesterone secretion by bovine granulosa cells and dispersed bovine luteal cells. These extracts produced a dose-dependent inhibition (23-92%) of progesterone synthesis by bovine granulosa cells. However, no inhibitory activity could be demonstrated in similarly prepared extracts from term placentae. Inhibitory activity could be extracted from both maternal caruncles and fetal cotyledons. In the presence of 2 mg/ml of maternal caruncle extract, basal progesterone secretion was dramatically reduced (90%), as was steroidogenesis in the presence of bovine lutenizing hormone (bLH) and 8 bromocyclic (Br)-cAMP. Moreover, coincubation of dispersed luteal cells and dispersed fetal or maternal placental cells from 100- to 150-day placentae produced a significant (50%) reduction in progesterone content of the medium. The addition of 2 mg/ml of caruncle or fetal cotyledon extract from 100- to 150-day placentae also produced 100% and 50% inhibitions, respectively, of progesterone secretion by dispersed placental cells. Thus, the inhibitory factor appears to be produced by cells of both the maternal and fetal placenta. It is heat-stable and not extractable by ether. The inhibitory substance eluted was two distinct peaks from Sephadex G-100 columns, one with a molecular weight of about 60,000 daltons and the other about 30,000 daltons. Using isoelectric focusing, several peaks of inhibitory activity were obtained, one with a pI of 3-5, the others having pIs between 6 and 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological and molecular characterization of plasminogen activator inhibitors 1 and 2 in baboon (Papio anubis) placental tissues

Two major plasminogen activator inhibitors (PAI-1 and PAI-2) increase in the peripheral circulation during pregnancy in humans. PAI-1 is of vascular endothelial origin whereas PAI-2 is produced primarily by human placental tissues. This study was undertaken to determine a) if PAI-1 and PAI-2 are also present in the baboon and b) their association with pregnancy. Citrated plasma was obtained from pregnant baboons sequentially at 15 +/- 3-day intervals between Days 30 and 140 of pregnancy. PAI activity increased significantly (p less than 0.05) at Day 120 (15.3 IU/ml) and 140 (21.8 IU/ml) of gestation and returned to baseline (2.6 IU/ml) 48 h post cesarean section. Placental tissues obtained at cesarean section during the third trimester were either placed in explant culture, fixed for immunocytochemistry, or frozen for RNA extraction. Western blot analysis of tissue culture media (TCM) indicated that the polyclonal antibody to PAI-1 reacted with a major band (Mr 47 000) in TCM from placental tissues while the PAI-2 antibody reacted primarily with a doublet (Mr 67 000 and 69 000) in these same media. PAI-1 was immunocytochemically localized primarily in the chorioamniotic tissue (CAM-D) and PAI-2 was found predominantly in placental villi. Slot blot hybridization with cDNAs to PAI-1 and PAI-2 indicated that the mRNA for PAI-2 was found primarily in placental villi, whereas the mRNA for PAI-1 was present in all three tissue compartments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proline metabolism in the conceptus: implications for fetal growth and development

Although there are published studies of proline biochemistry and nutrition in cultured cells and postnatal animals, little is known about proline metabolism and function in the conceptus (embryo/fetus, associated placental membranes, and fetal fluids). Because of the invasive nature of biochemical research on placental and fetal growth, animal models are often used to test hypotheses of biological importance. Recent evidence from studies with pigs and sheep shows that proline is a major substrate for polyamine synthesis via proline oxidase, ornithine aminotransferase, and ornithine decarboxylase in placentae. Both porcine and ovine placentae have a high capacity for proline catabolism and polyamine production. In addition, allantoic and amniotic fluids contain enzymes to convert proline into ornithine, which is delivered through the circulation to placental tissues. There is exquisite metabolic coordination among integrated pathways that support highest rates of polyamine synthesis and concentrations in placentae during early gestation when placental growth is most rapid. Interestingly, reduced placental and fetal growth are associated with reductions in placental proline transport, proline oxidase activity, and concentrations of polyamines in gestating dams with either naturally occurring or malnutrition-induced growth retardation. Conversely, increasing proline availability in maternal plasma through nutritional or pharmacological modulation in pigs and sheep enhances concentrations of proline and polyamines in placentae and fetal fluids, as well as fetal growth. These novel findings suggest an important role for proline in conceptus metabolism, growth and development, as well as a potential treatment for intrauterine growth restriction, which is a significant problem in both human medicine and animal agriculture.     

Immunohistochemical localization of chorionic gonadotrophin on baboon placenta, dispersed trophoblast cells and those derived from blastocysts grown in vitro

An immunohistochemical technique using a high specificity antiserum against baboon CG was used to demonstrate the presence of a CG-like material on: (1) fixed baboon placental sections collected between 31 and 39 days of gestation, (2) trophoblast monolayers derived from hatched embryos grown in vitro for 15 days and (3) trophoblast cells derived from cells dispersed from placentae collected between Days 31 and 39 of pregnancy. A specific radioimmunoassay was used to detect concentrations of baboon CG in daily spent medium. Immunohistochemical studies showed that material cross-reacting with CG was present on all the three sources of trophoblast. The embryos secreted CG from attachment onwards and immunoactive CG was measurable in daily spent medium collected from placenta-derived trophoblast cultures. It is concluded that baboon CG is localized in the syncytiotrophoblast of fixed placental sections and cellular trophoblast derived from cultured embryos and placental cells.     

Ultrastructural studies on the boundary tissue of the seminiferous tubules of different mammals

The aims of our studies were to compare the ultrastructure of the boundary tissue of seminiferous tubules of various mammals (rat, mouse, hamster, guinea pig, rabbit, ram, bull and man). Visual analysis of electron micrographs revealed the similarity of structure of all layers at investigated animals. The boundary tissue consists of 4 layers: 1) amorphous inner lamina, 2) cellular inner lamina, 3) amorphous outer lamina, 4) cellular outer lamina. The outer lamina of boundary tissue of rat, mouse and hamster revealed in histochemical reactions meshes resembling honey-combs. The wall of seminiferous canalicules of bull and ram consists of more bigger and different structure than one at the other laboratory animals. The most different structure of boundary tissue in man was observed. The capillary vessels penetrate in the myofibroblastic layer, when comparted to that found in other mammals on the surface of the wall.     

Purification and N-terminal characterization of Chinchilla villidera alpha-1-antitrypsin

1. Chinchilla, Chinchilla villidera, alpha-1-antitrypsin has been purified to homogeneity and partially characterized according to mol. wt, amino acid and carbohydrate composition and N-terminal amino acid sequence (30 residues). 2. The mol. wt is between 52,000 and 55,000 as determined by PAGE or sedimentation equilibrium. 3. The best alignment between chinchilla, human and baboon alpha-1-antitrypsin amino acid sequences offsets the chinchilla sequence 6 positions vs the primate structures. 4. This alignment suggests potential importance of the sequence His-Glu-Gln-Glu-His at positions 11-15. 5. Additionally, the segment Leu-Ala-Glu-Phe-Ala, positions 25-29, is strictly conserved. 6. Shorter N-terminal sequences available for rat and rabbit alpha-1-antitrypsin appear to follow the offset alignment vs the primate structures.     

Comparative histochemical distribution of glycogen and alkaline phosphatases in the placenta

Summary The distribution of glycogen, non-specific alkaline phosphatase, and specific phosphatases acting on adenosine monophosphate, adenosine triphosphate, inosine triphosphate, thiamine pyrophosphate, uridine diphosphate, fructose-6-phosphate, fructose-1:6-diphosphate, and glucose-6-phosphate is described in the placentae and accessory structures of the horse, sheep, cat, dog, ferret, rat, rabbit, guinea pig, and man, and in the yolk-sac of the chick, the oviviparous fish Limia maculata, and man.Correlation between the distribution of these substances and placental function is sought, and the results are discussed with respect to the trophoblast, decidua, giant cells, yolk-sac, non-placental chorion and maternal epithelium and uterine secretion, and allantois. The significance of the presence of the enzymes in the carnivore interstitial matrix, the ferret thickened endoderm and the rodent spongy zone is also considered.