Sequential isolation of lamellar bodies and surfactant fractions from rat lungs.
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Animals↗
Biomedical subjects
A R Beaudoin
Publications and source records attributed to A R Beaudoin.
The origin of the zymogen granule membrane of the pancreatic acinar cell as examined by ultrastructural cytochemistry of acid phosphatase, thiamine pyrophosphatase, and ATP-diphosphohydrolase activities.
Cytochemical distributions of acid phosphatase, thiamine pyrophosphatase, and ATP-diphosphohydrolase activities have been examined on thin sections of rat pancreas and on isolated zymogen-granule membranes. Acid phosphatase was found in the rigid lamellae separated from the Golgi stacked cisternae, in condensing vacuoles, and in the trans-saccules of Golgi apparatus; it was not detected in purified zymogen-granule membranes. Thiamine pyrophosphatase was detected in trans-saccules of the Golgi apparatus, in purified zymogen-granule membranes, and in the plasmalemma of the acinar cell. It was absent in condensing vacuoles. The ATP-diphosphohydrolase activity has a distribution similar to thiamine pyrophosphatase. These observations illustrate the similarity between the trans-saccules of the Golgi apparatus and the membrane of mature zymogen granules and the disparity between the latter membrane and the membrane of the condensing vacuole. They suggest that the condensing vacuole might not be the immediate precursor of the zymogen granule as commonly assumed. An alternative possibility would be that condensing vacuoles would fuse with the trans-saccule (transition) of the Golgi apparatus which in turn would form mature zymogen granules.
Acid Phosphatase↗
Teratogenic action of platinum thymine blue.
The teratogenic activity of the antitumor agent cisplatinum-2-thymine (platinum thymine blue) was investigated in rats. Pregnant Wistar-derived albino rats were given single ip injections of an aqueous solution of platinum thymine blue (PTB) at one day of pregnancy from day 5 through day 14 (sperm day=day 0). The dosages used ranged from 20 to 80 mg/kg maternal body weight. At autopsy (day 20) fetuses were recovered and subsequently examined for skeletal and soft-tissue abnormalities. PTB was embryolethal and teratogenic at several stages during rat gestation. Embryonic death occurred following all doses, and was dose dependent, except at day 5. The majority of malformed fetuses, however, were observed only after treatment at day 6 or 7 following injection with 50, 60, or 80 mg/kg. Eye defects were the predominant abnormality followed by hydrocephalus, gastroschisis, and ectopia cordis. The skeleton was only slightly affected. PTB is a potent inhibitor of DNA synthesis, but its mechanism of teratogenic action is unknown.
Abnormalities, Drug-Induced↗
Newly synthesized secretory proteins from pig pancreas are not released from a homogeneous granule compartment.
The pancreatic secretion of anesthetized pigs was collected by cannulation after pulse labeling with [3H]leucine. Collection at 5 min intervals started immediately post-pulse labeling up to 85 min. The volume, the protein content and the trichloroacetic acid-precipitable radioactivity of the juice were measured. The specific radioactivity of the secretory proteins was compared to that of a zymogen granule fraction isolated from the same animal. The latter was very much higher. Caerulein stimulation for 5 min at 80 min post-pulse caused a sharp drop in the specific activity of secretory proteins in the juice, to a level lower than that of the zymogen granule content. These data support the concept of more than one pool of secretory proteins in the pancreas and are incompatible with the concept that secretory proteins derive from an homogeneous granule compartment in a functionally homogeneous population of cells. To explain our results the hypothesis of a second intracellular route for the secretory proteins in proposed.
Animals↗
Kinetic effects of Ca2+ and Mg2+ on ATP hydrolysis by the purified ATP diphosphohydrolase.
ATP diphosphohydrolase (EC 3.6.1.5) catalyzes the hydrolysis of diphospho- and triphosphonucleosides and is sensitive to divalent cations. In this paper, we investigated the dependence of ATP hydrolysis on the concentration of free Mg2+ and Ca2+ and the cation ATP complexes. The enzyme was isolated from porcine zymogen granule membranes, solubilized in Triton X-100, and purified on a 5'-AMP-Sepharose 4B affinity column resulting in a 1500-fold purification. Free unprotonated ATP4- was hydrolyzed in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. When hydrolysis rate was measured at different concentrations of the cation-ATP complex at constant free cation concentrations, normal hyperbolic curves were obtained. In CaCl2, both Kapp and Vapp increased as free Ca2+ increased from 25 to 1000 microM. In MgCl2, Kapp increased and Vapp decreased as free Mg2+ increased from 25 to 500 microM. From the rapid equilibrium rate equation, Ks and Vmax values of the substrates were calculated. We found that free ATP4-, Ca-ATP2-, and Mg-ATP2- are substrates and free cations do not bind the enzyme.
Adenosine Triphosphate↗
Immunocytochemical localization of actin in the pancreatic exocrine cell.
Immunoreactive actin molecules have been localized in pancreatic acinar cells by the protein A-gold technique. The labeling was found at the level of the filamentous cell web, and in close association with the Golgi cisternae, condensing vacuoles, and zymogen granules delimiting membranes as well as with the plasma membrane. A weak labeling was also present over the dense content of the zymogen granules. The association of actin with different membranes implicates that contractile proteins might constitute structural membrane proteins and, thus, might play an important role in protein secretion.
Actins↗
Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins.
A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.
Animals↗
An in vivo/in vitro evaluation of teratogenic action.
Several compounds were administered to pregnant Wistar-derived rats either 24 or four hours prior to the recovery of day 10 embryos for in vitro culture in Waymouth's medium and fetal calf serum. The compounds tested were 2-amino-1,3,4-thiadiazole (thiadiazole), cadmium sulfate, 1,2-dibromo-2,2-dichloroethyl dimethyl phosphate (dibrom), 2-(sec-Butyl)-4,6-dinitrophenol (dinoseb), led nitrate, polybrominated biphenyls (PBB), sodium arsenate, and trypan blue. After 24 hours in culture, two thirds of the embryos were recovered for examination. The remaining one third were continued in culture until 42 hours. Recovered embryos were examined for rotation of the embryonic axis, heart rate, establishment of the visceral yolk sac circulation, somite number, growth of the limb buds, closure of the neural tube, and development of the allantois and amnion. All tested compounds inhibited the rate of development in vitro.
2,4-Dinitrophenol↗
The failure of glutamic acid to protect the rat embryo against the action of trypan blue.
The effect of L-glutamic acid on the embryolethal and teratogenic action of trypan blue was investigated in Wistar albino rats. L-glutamic acid was either incorporated into the diet, from gestation day 2 to day 20, or suspended in sesame oil and administered by gavage, from gestation day 6 to day 10. The day of finding sperm in the vaginal smear was designated day 0 of pregnancy. A teratogenic dose of trypan blue was injected at day 8 of pregnancy, either intraperitoneally (14 mg/kg maternal body weight) or subcutaneously (160 mg/kg). The amount of glutamic acid consumed, after the injection of trypan blue, ranged from 600 to 1,500 mg/rat/day. Pregnancy was terminated at day 20, and the fetuses were recovered and examined. Glutamic acid failed consistently to protect the rat embryo against the lethal and teratogenic action of trypan blue. These results are in contrast to those obtained in mice. the administration of sesame oil alone was found to cause embryonic death but not malformations.
Abnormalities, Drug-Induced↗
An exploration of the role of hydroxyurea injection time in fetal growth and teratogenesis in rats.
Pregnant Wistar rats were injected with hydroxyurea (HU) intraperitoneally (IP) t one of several 6-hour intervals on days 9-10.75 to study the role of circadian growth variations in the teratologic response of the fetuses. Two stocks of rats were studied and the results in each compared. The fetal response to HU, as observed at day 21, was not detectably modified by circadian fetal growth variation. No correlations between hour of HU administration and fetal weight, placental weight, resorption, or total malformation rates were found. Cyclic variations in the incidence of hydronephrosis and left umbilical artery was observed, but it was not clear that these were related to maternal light:dark cycles. Difference of response between two stocks of rats included marked variation in the incidence and type of malformations and variations in the timing of peak incidence for some but not all malformations.
Abnormalities, Drug-Induced↗
Cytological effects of urecholine stimulation on the rat pancreas.
Stimulation of the exocrine pancreas by the secretagogue urecholine caused degranulation of the acinar cells. Under in vivo conditions, this degranulation is not uniform throughout the tissue. Indeed some of the acini are almost completely depleted of their granules while others display the appearance of resting acini. A noticeable feature is that all the cells of the same acinus display a comparable degree of degranulation. Moreover, groups of neighbouring acini seem to respond simultaneously suggesting that the secretory stimulus is propagated from one acinus to the other. In vitro stimulation of dispersed acini also showed that some of the acini are more responsive than others indication that this phenomenon can not be attributed to accessibility of the secretagogue to its receptor. These observations lead us to accessibility of the secretagogue to its receptor. These observations lead us to the concept that the response of the pancreatic acinar cell is controlled at the level of the acinus.
Animals↗
Distribution of amylase and chymotrypsin in four regions of the rat pancreas.
Recent observations have confirmed the existence of more than one pool of secretory proteins in the rat pancreatic tissue. To determine if these different pools could be located in the different regions of the rat pancreas, the amylase and chymotrypsin contents have been measured in the biliary, duodenal, gastric, and splenic regions. On a tissue DNA content, protein content, or a fresh weight basis, the proportions of these two enzymes are comparable in the four regions. It is therefore postulated that heterogeneity of enzyme composition exists either within these regions or within the acinar cell itself.
Amylases↗
Anesthetics as teratogens: nitrous oxide is fetotoxic, xenon is not.
Exposure of pregnant rats to the anesthetic nitrous oxide on the ninth day of gestation causes fetal resorption, skeletal anomalies, and macroscopic lesions including encephalocele, anophthalmia, microphthalmia, and gastroschisis. The inert gas xenon, which has anesthetic properties similar to those of nitrous oxide, does not cause teratogenic effects under the same experimental conditions.
Anesthetics↗
Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas.
An ATP diphosphohydrolase (EC 3.6.1.5) from the pancreas of the pig has been characterized and purified. The enzyme which has an optimum pH between 8 and 9 is specific for diphospho- and triphosphonucleosides. The Km values for ADP and ATP are 7.4 and 7.3 x 10(-4) M, respectively, and the purified enzyme has specific activities of 13 and 15.2 mumol of Pi/min/m of protein, respectively. It requires calcium or magnesium ions and it is insensitive to ATPase inhibitors, namely oligomycin, ouabain, and ruthenium red, and to levamisole, an inhibitor of alkaline phosphatase. Denaturation experiments, by heat and trypsin treatments, indicated that only one enzyme is involved. This is confirmed by the solubilization and purification process and by polyacrylamide gel electrophoresis. A 270-fold purification was obtained by centrifugation and successive column chromatography on Sepharose 4B and Affi-Gel blue. It is a glycoprotein with a molecular weight of 65,000 as estimated by polyacrylamide gel electrophoresis.
Animals↗
A study of cartilaginous fusions, DNA synthesis, and protein synthesis in embryonic rat limbs after injection of aminothiadiazole.
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Abnormalities, Drug-Induced↗
Exocytosis in mammalian cells. VI. Temperature dependence of pancreatic exocrine secretion.
The effects of temperature on amylase secretion in vitro have been measured under stimulated and nonstimulated conditions. In both cases a very rapid decrease of secretory rate was observed between 37 and 25 degrees C (60-70%). The decrease is less pronounced between 25 an 15 degrees C. Between 15 and 13 degrees C a drop of secretory activity (10%) was observed under stimulation whereas an increase was measured in absence of stimulus. At lower temperatures the rate of secretion falls again abruptly. These results show that the secretory pattern of amylase from pancreas in vitro as a function of temperature is very different under stimulated and nonstimulated conditions. We suggest that the abrupt change of secretory activity between 15 and 13 degrees C could correspond to a change of physical state of the membranes involved in the exocytosis process.
Amylases↗
Fetal and placental responses to artificially induced hyperthermia in rats.
Pregnant rats were utilized to study the effect of maternal hyperthermia on fetal development. Eight groups of six to eight rats were exposed to ambient temperatures of 43-44 degrees C at various stages of pregnancy. All rats were killed on day 20 of gestation. Edema, microencephaly and microphthalmia followed heat treatment on day 4, 6, or 8 and skeletal defects occurred on day 10 of gestation. Apparently heat stress of dams after day 14 of gestation had little or no effect on embryos. Most placentas from day 6-10 treatment groups were significantly heavier than control and exhibited extensive thickening and necrosis of decidua basalis. Our results suggest that the rat is a useful model for investigating maternal hyperthermia as a possible cause of human placentophathies and fetal retardation.
Animals↗
A convenient method for the ATPase assay.
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Adenine Nucleotides↗