L-gulonolactone oxidase activity and vitamin C status in riboflavin-deficient rats

The effect of riboflavin deficiency on the activity of L-gulonolactone oxidase [L-gulono-γ-lactone : oxygen 2-oxidoreductase, EC 1.1.3.8] and on vitamin C status was studied. A marked decrease in the specific activity of L-gulonolactone oxidase was observed in the liver microsomes isolated from riboflavin-deficient rats: the specific activity was approx. one-third of that in the microsomes isolated from control rats. The L-ascorbic acid content in the liver of the riboflavin-deficient rats was approx. one-half of that in the liver of the control rats. It seems that the rate of production of L-ascorbic acid in the riboflavin-deficient rats is limited by the decreased level of L-gulonolactone oxidase activity. Immunotitration using rabbit antiserum directed to L-gulonolactone oxidase revealed that a substantial amount of an inactive form of this enzyme is present in the liver microsomes of the riboflavin-deficient rats. L-Gulonolactone oxidase activity in the microsomes of these rats increased by approx. 35% upon addition of FAD, but it was slightly decreased by the addition of FMN or riboflavin. These results indicate that the liver microsomes of the riboflavin-deficient rats contain a protein which exhibits L-gulonolactone oxidase activity upon addition of FAD.     

Treatment of a metabolic disease, scurvy, by administration of the missing enzyme

Reaction of immunoprecipitated L-gulonolactone oxidase with glutaraldehyde allows multiple administrations of large amounts of this enzyme extracted from either chicken or rats to guinea pigs. L-Gulonolactone oxidase converts L-gulonolactone to ascorbic acid, and its absence from guinea pigs and primates results in their requirement for this vitamin. By administration of this enzyme guinea pigs are able to survive on an ascorbic-acid-deficient regimen.     

Developmental Changes in Rat Liver Xanthine Oxidase(s) and Its Intracellular Distribution

Developmental change and subcellular distribution of xanthine oxidase in the rat liver were examined.

The specific activity of the fetal liver xanthine oxidase increased sharply to the levels of the adult liver on the day of the birth. After birth, the activity dropped rapidly and on the 14th day after birth it was about 1/4 of adult level. Then the activity was regained and around 28th day after birth it was about the same as in adult level.

In the livers from 80 days old rats, about 60% of total xanthine oxidase activity was found in soluble fraction and the rest was distributed among particulate fractions including microsomal, lysosomal, mitochondrial and nuclear fractions.

In contrast to the adult livers 80% of total xanthine oxidase activity in fetal liver was found to be in particulate fractions.

From kinetic studies of xanthine oxidases in particulate and soluble fractions it was suggested that xanthine oxidase in soluble fraction and xanthine oxidase in particulate fraction might be different in their natures of protein molecule.     

Developmental changes in vitamin A level and lack of retinyl palmitate in chick lungs

1. Developmental changes in retinol and retinyl palmitate contents in lungs of chick embryos and posthatch chicks were investigated. 2. Remarkable changes in the lung retinol levels were found during development of chicks. Embryonic lungs 5 days prior to hatching contained the highest content of retinol. The level then declined rapidly and was lowest on 1 day before hatching. 3. Its level then rose substantially within 7 days after hatching. 4. No retinyl palmitate in chick lungs was detectable at any of the developmental stages examined, nor even in adult hen. 5. Serum retinol level changed in parallel with the lung retinol. 6. The patterns of changes in liver retinol and retinyl palmitate were remarkably different from that occurring in the lung retinol. In chick embryonic livers, the levels of them were low, followed by a rapid increase after hatching. 7. The high level and its rapid decrease of lung retinol content during development of chick embryos may be functionally connected with retinol action in embryonic lungs for cellular differentiation and maturation.     

Rat liver and intestinal mucosa differ in the developmental pattern and hormonal regulation of carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase gene expression

cDNA probes were employed to measure levels of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyltransferase (OCT) mRNAs in fetal and neonatal livers and intestines. In the fetal liver, significant levels of OCT mRNA were present at 15-days gestation while CPS mRNA could not be detected until day 17 of fetal development. Apart from a small decline just after birth, amounts of both mRNAs increased steadily to reach adult levels in postnatal life. In contrast to the situation in liver, CPS and OCT mRNA levels in the fetal intestine rose rapidly to peak at day 21 of gestation and then declined steadily in the first seven days after birth. Using the methyl-sensitive restriction isoschizomeric pair, MspI/HpaII, the 5' ends of both the CPS and OCT genes were shown to undergo demethylation during development. In the case of the OCT gene, however, the hypomethylation characteristic of the adult liver and intestinal mucosa was not observed in the 15-day-old fetal liver, where significant levels of gene expression had already been established. Levels of CPS and OCT mRNA in livers of adults responded to glucagon in normal animals (1.5-fold and 2.2-fold increases, respectively) and to dexamethasone in experimentally induced diabetic animals (3-fold increase in CPS mRNA with no change in OCT mRNA). These treatments were all without effect on the levels of CPS and OCT mRNA in intestinal mucosa.     

Ascorbic acid and L-gulonolactone oxidase in lagomorphs.

1. The activity of L-gulonolactone oxidase (EC 1.1.3.8) in the liver of eastern cottontail rabbits (Sylvilagus floridanus) is about 10-fold greater in winter than in summer. 2. L-gulonolactone oxidase activity is low and tissue ascorbate high during all seasons in snowshoe hares (Lepus americanus). 3. Liver contents of ascorbate fall to low levels in L. americanus fed on rabbit chow in the laboratory. 4. The activity of L-gulonolactone oxidase in liver of Sylvilagus and Oryctolagus is depressed by feeding high levels of L-ascorbic acid. 5. The New Zealand White breed of domestic rabbit (Oryctolagus cuniculus) has considerably higher levels of L-gulonolactone oxidase and liver ascorbate than does the Dutch breed. 6. In a wild population of Oryctolagus sampled in Australia L-gulonolactone oxidase levels were intermediate between those of the two domestic breeds and more variable than either.     

Arginase activity patterns and their regulation during embryonic development in liver and kidney

The developmental patterns for mouse liver and kidney arginase were measured by a sensitive radioactive assay from day 8 of gestation until adulthood. On day 8 high arginase activity is generally distributed throughout early embryos. Then, as development proceeds, the arginase activity drops rapidly in liver and kidney, apparently because of mass increase unaccompanied by net arginase synthesis. Suddenly, on day 12 of gestation in liver and on day 16 in kidney, arginase activity begins to accelerate toward adult values.In order to study the mechanisms controlling arginase acceleration, 12- and 13-day fetal livers were explanted to organ cultures containing various exogenous chemicals, and subsequently assayed for arginase. Physiological concentrations of hydrocortisone causes the arginase activity to rise more than 100-fold to adult levels within 4 days in culture. Glucagon, thyroxine, and dibutyryl adenosine-3′-5′-cyclic phosphate have no effect in this system. Experiments with cycloheximide, actinomycin D, and 5-fluorodeoxyuridine suggest that the hydrocortisone response is dependent upon protein and RNA synthesis but independent of DNA synthesis.     

Antithrombin III mRNA in adult rat liver and kidney and in rat liver during development

Using Northern blots the size of antithrombin III (AT III) mRNA in rat liver was found to be 1650 nucleotides. Adult rat kidney also contained a slightly smaller mRNA at about 20% the level in liver. The ontogeny of AT III mRNA in the liver was assessed by dot blot hybridization. The mRNA was detectable at the earliest age examined (14th day of gestation) at about 15% of the adult levels. After the 17th day of gestation the levels of antithrombin III mRNA rise reaching 50% of adult levels at birth. After birth the mRNA levels rise to 75% of adult levels by the 5th day and reach adult levels by 40 days after birth. We suggest that foetal AT III is produced by both the foetal liver and by placental transfer of the maternal inhibitor.     

Polyamines and diamine oxidase activity in maternal, embryonal, and fetal tissues of rat after chronic ethanol consumption

The effects of maternal ethanol consumption for 4 weeks before and throughout gestation on polyamine content and diamine oxidase activity of maternal, embryonal and fetal tissues are reported. At the 12th day of pregnancy, a decrease of putrescine in the liver of the mother and marked increases in putrescine, cadaverine and spermidine in embryos were observed. At day 18, putrescine and cadaverine diminished in maternal liver and placenta, and no changes in amine content in fetal liver and brain were found. At day 12, diamine oxidase activity increased in maternal liver and placenta, whereas it greatly diminished in embryos. At day 18, enzyme activity decreased in maternal liver, placenta, fetal liver and brain. These results indicate that chronic ethanol ingestion induces alterations in polyamine concentrations and metabolism in growing and developing tissues during pregnancy that might contribute to the adverse effect of ethanol on conceptual development.     

ONTOGENY OF CHICKEN HEMOGLOBIN. III. IMMUNOLOGICAL STUDY OF THE HETEROGENEITY OF HEMOGLOBIN IN DEVELOPMENT

By applying the double diffusion technique of Ouchterlony and the immunoelectrophoresis, sequence in the appearance of antigens reactive with antisera against HbCOs from 5-day embryos and adult chickens, and the major component of adult HbCO in the course of chicken development has been studied.
Antigenic components reacting to antiserum against HbCO from 5-day embryos have been detected throughout development. Three globin-like components specific to early embryos are detectable in embryos to 2 days of incubation. Two Hb components are detectable in embryos from 3 to 5 days of incubation; one is apparently specific to embryos, while the other seems to be somewhat different from adult Hb. After 6 days of incubation only one adult Hb component is detectable.
Antigenic components reacting to antiserum against HbCO from adults have also been detectable throughout embryonic life. One globin-like component specific to early embryos can be found in embryos to 2 days of incubation. One or two Hb components which are probably specific to embryos can be detected in embryos from 3 to 5 days of incubation. After 6 days of incubation one adult Hb component is detectable, while one globin-like component specific to adults can be found after 15 days of incubation. Further, the other globin-like component is detectable after 3 days of incubation.
Antigenic components reacting to antiserum against the major component of adult HbCO have been detected throughout development; one which seems to be somewhat different from either adult Hb components can be found in embryos from 2 to 5 days of incubation, while the other which is identical with the major component of adult Hb is detectable after 6 days of incubation.     

Teratogenic effects of dilantin on thoraco-abdominal organs of developing chick embryos

Congenital anomalies on some viscera like heart, liver and kidney have been investigated in chick embryos after a single injection of dilantin (3 mg/egg), a known antiepileptic drug, on 4th day of incubation. On 19th day of incubation, chick embryos were collected to observe the gross malformations and histological changes in heart, liver and kidney. On gross examination, visceroptosis (29%), thin anterior abdominal wall (28%), ectopia cordis (10%) and dextrocardia (1%) were observed. Histological examination of the kidney revealed glomerular degeneration in kidney while in liver, dilated central veins with degenerated hepatocytes were present. Longitudinal section of the heart showed thicker musculature specially of ventricles with a narrower lumen in comparison to that of the control. The results indicate teratogenicity of dilantin in developing chick embryos.     

Acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of pre and post hatched chicks

1. The behaviour of total acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of chick embryos between 11th and 21st day of development and of 8 and 180-day-old chicks is described. 2. Total acid soluble carnitine fluctuates around the same levels in the brain, liver and muscle until 18th day of development, whereas it attains a peak on that day in the heart. At hatching compared to 18th day, it suddenly increases three times in the muscle, drops not significantly in the heart and brain, but sharply in the liver (-40%). However the levels are always higher than those of the grown chick in the brain but lower in the other tissues. 3. Free carnitine levels are almost constant in all tissues during the embryonic life; if compared to adult ones, they are very much lower in the liver, heart and muscle, but higher in the brain, even in 8 day-old chick. 4. Short chain esterified, carnitine reaches a maximum on 18th day of egg incubation in the liver, brain and heart; in the muscle it stays on constant levels until this day and then rapidly increases so that at hatching it doubles the values. 5. The short chain esterified to free carnitine percentage ratio peaks in all tissues on 18th day of development, attaining figures which are well above those determined in the grown chick.     

Changes of gamma-glutamyltranspeptidase activity in the rat during development and comparison of the fetal liver, placental and adult liver enzymes

Changes in the activity of gamma-glutamyltranspeptidase (GGT, EC 2.3.2.2) during development in rats were investigated. The activity of GGT in fetal liver increased rapidly immediately before birth, reached a maximum at birth and then decreased rapidly within a week after birth to nearly the level in adult rat liver. In contrast, placental GGT showed higher activity at an early stage (from day 13 to day 15) of the gestation period, but the activity decreased in the last part of fetal life. The activity in the amniotic fluid increased significantly just before birth, in parallel with the increase of activity in the fetal liver. No change of activity in the uterine wall was observed throughout gestation. The kinetic and immunological properties of partially purified GGTs from fetal liver and placenta were almost identical with those of adult liver GGT. However, the activity of soluble GGT in fetal liver was less effectively inhibited by antibody against adult kidney GGT. Thus, it is likely that at least one isozyme of GGT is present in the soluble fraction of fetal liver.     

Pyridoxamine (pyridoxine) phosphate oxidase activity in mammalian tissues

1. Vitamin B6-sufficient rats had moderate pyridoxamine-P oxidase specific activities in heart, brain, kidney and liver, but no detectable activity in skeletal muscle. Vitamin B6-deficiency in rats resulted in a decreased oxidase activity in liver but no change in the activities in other tissues. 2. The pyridoxamine-P oxidase activity in vitamin B6-sufficient mice was high in liver, moderate in brain and kidney, and not measurable in skeletal muscle and heart. Vitamin B6-deficient, compared with control mice, had decreased oxidase activities in brain, kidney and liver. 3. Mouse erythrocytes took up pyridoxine more rapidly than did rat and human erythrocytes. 4. Mouse and human erythrocytes rapidly converted pyridoxine to pyridoxal-P. Rat, hamster and rabbit erythrocytes had appreciably lower pyridoxamine-P oxidase activity than did mouse and human erythrocytes.     

Studies on cation-induced membrane vesicle aggregation of porcine intestinal brush borders

Protein alterations during the development of the mouse brain were studied by two-dimensional gel electrophoresis. A protein spot with a molecular weight (MW) of 68,000 and pI value of 5.6 was found in the brain of the 11th day of gestation. Between the 12th and the 14th day of gestation, spots with the same MW and lower pI values appeared progressively. Neuraminidase digestion converted the pI of these acidic spots to 5.6. Thus, increased sialylation appeared to occur during this period. This class of molecules became hardly detectable on the 15th day, and disappeared completely after the 16th day. Analogous spots were present in the heart, liver, and stomach of the embryos, although the increased sialylation was not observed in the liver. No adult organs so far examined showed these spots. On the other hand, two polypeptides (MW 55,000, pI 4.7, and 53,000, pI 4.6) appeared in the brain on the 13th day of gestation and persisted throughout the fetal period. After birth, they became hardly detectable. Furthermore, a spot (MW 48,000, pI 4.8) became newly detectable in the brain 4-5 weeks after the birth.     

Changes in polyamine-oxidizing capacity of peroxisomes under various physiological conditions in rats

Rat liver peroxisomal polyamine oxidase activity was determined under various physiological conditions by using the peroxidase method with phenol and 4-aminoantipyrine. N1-Acetylpolyamines such as N1-acetylspermine and N1-acetylspermidine were better substrates than the free polyamines. The polyamine oxidase activity in rat peroxisomes increased significantly when cell proliferation was high. The activity began to appear in fetal liver at the 16th approximately 18th day of pregnancy and peaked in neonatal liver on the first day (approx. 1.7-times higher than in adult liver). In regenerating rat liver, only polyamine oxidase activity among the peroxisomal enzymes tested was increased considerably 12 h after partial hepatectomy (approx. 2.8-fold over the control liver). Finally, the enzyme activity was significantly increased by administration of clofibrate, a peroxisome proliferator, which also causes hepatomegaly. In all cases, the increase in polyamine oxidase activity was not more than 3-fold. Since the level of polyamine oxidase activity in the normal liver is more than adequate in relation to the level of the substrates, the slight but significant increase under conditions of cell proliferation may have a role in modulating levels of polyamines in the proliferating liver tissue.     

Comparison of plasma ACTH and corticosterone levels after embryo bursectomy

Plasma levels of both adrenocorticotropic hormone (ACTH) and corticosterone (B) were determined in embryos (day 15 of incubation), chicks (day 3 after hatch) and young chickens (8 weeks). Experimental animals were bursectomized at 80 hr of incubation, i.e., before any anlage of the bursa of Fabricius could develop. Bursectomized (BFX) animals were compared to sham-operated controls (T), in basal, resting condition and 7 (ACTH) or 14 min (B) after ether stress was delivered for 30 sec. Basal B and ACTH levels seemed not to be significantly modified in BFX embryos, chicks and chickens. Hypophysial and adrenocortical response to stress appeared more precociously in BFX embryos (day 15 of incubation) than in intact ones (day 19). The non stress-responsive period that was observed for one week after hatch of T birds did not appear in 3-day-old BFX chicks whose both B and ACTH stress-induced levels were as high as in intact adults. In contrast, adrenocortical and pituitary corticotropic responses to stress were markedly impaired (by 50%) in adult BFX chickens as compared to intact controls.     

Hexokinase isoenzymes in tissues of the adult and developing guinea pig

Changes in the activities and isoenzyme distribution of hexokinase were determined in a number of tissues during the development of the guinea pig. The total activity in the fetal liver showed a large fall during the second half of gestation to reach adult values by term. With normal diet the fetal, neonatal, and adult livers had isoenzymes I and III but little or no detectable IV (glucokinase). The fetal liver had predominantly type I, but the proportion of type III increased during development. The kinetics of the guinea pig isoenzymes were similar to those reported for the rat. Two additional isoenzymes with mobility between I and II were detected in the fetal liver and blood. They appear to have kinetic properties similar to type I. Detectable liver glucokinase activity was induced by glucose administration to adult guinea pigs. The total activity in kidney, brain and skeletal muscle showed a postnatal rise while in the fetal heart it was high and declined after birth. These tissues contained predominantly type I with varying proportions of type III hexokinase. The ratio of particulate-bound to soluble hexokinase varied from tissue to tissue. All except the liver showed a significant increase in binding after birth. The changes are discussed in relation to the control of glucose utilization in the fetal and neonatal periods.     

The oxidation of choline by liver slices and mitochondria during liver development in the rat

Betaine is the major oxidation product of [Me-¹⁴C] choline produced by rat liver slices. Liver slices from adult rats rapidly oxidize [Me-¹⁴C] choline to betaine and the bulk of the betaine produced is recovered in the incubation medium. Considerably more choline is oxidized to betaine than is phosphorylated to phosphorylcholine. The rate of phosphorylation of choline appears to be independent of the rate of choline oxidation. Liver slices from fetal and young rats oxidize choline to betaine at a lower rate than adult liver slices.The ability of mitochondria to oxidize [Me-¹⁴C] choline to betaine aldehyde and betaine is considerably lower in fetal liver than in adult liver. The major product with both fetal and adult mitochondria is betaine aldehyde. Choline oxidation by mitochondria begins to increase 1 day prior to birth and increases progressively to adult levels by 18 days. The developmental pattern for choline oxidation is similar to the pattern for succinic dehydrogenase activity.