Developmental changes of chicken liver AMP deaminase.

The AMP deaminase activity measured in crude chicken liver extract did not change significantly during development. The livers of 10- and 14-day chick embryos, 1-day, 5-, 10- and 16-week-old chickens and adult hens were examined for the existence of multiple forms of AMP deaminase. Phosphocellulose column chromatography revealed the existence of two peaks of enzyme activity in the liver of 10- and 16-week-old chickens and adult hens. Kinetic studies with the preparations of AMP deaminase revealed sigmoid-shaped substrate-saturation curves at all developmental stages and hyperbolic-shaped saturation curves for the enzyme form appearing in 10-week-old chickens. All AMP deaminases investigated were susceptible to activation by ATP and inhibition by Pi. Kinetic and regulatory properties as well as pH optima of all the enzyme preparations tested indicate that AMP deaminase isolated from the embryos and from 1-day-old chicks was similar to the form I isolated from adult hens and differed significantly from the form II of this enzyme.     

Ontogenesis of the insulin receptor in the rabbit brain

We delineated the ontogeny of the brain insulin binding, insulin receptor number and affinity using plasma membranes isolated from the rabbit. Specific 125I-insulin binding and receptor number expressed per milligram of protein increased from the 20 day gestation fetus to the 1-day-old newborn, declining thereafter to attain adult values by day 6 of postnatal life. Specific 125I-insulin binding and the receptor number in the adult brain was less than the fetal and neonatal (1 day) brain receptors. Although a similar trend was observed specifically during fetal development, the changes in receptor number expressed per microgram DNA were not significant in the neonatal period. The adult brain insulin receptor number was higher than the 20- to 27-day fetus and similar to that of the 30-day fetus and the 1- to 5-day newborns. The total receptor number correlated linearly with the brain plasma membrane protein increment velocity. The affinity of the receptors increased during early fetal development (20-27 days) and remained constant thereafter in the postnatal period. We conclude that the ontogenic changes of the brain insulin receptors are similar to the ontogenic changes of brain plasma membrane protein. The developmental changes are more pronounced when the receptor number is expressed per milligram protein versus microgram DNA.     

NUCLEAR PROTEINS IN BRAIN OF 7-DAY-OLD AND ADULT RATS

Abstract— The incorporation of radioactive leucine into proteins of rat brain was considerably higher in the 7-day-old than in the adult rat. The greatest difference in the rate of protein synthesis between these two stages of development occurs in the mitochondrial fraction. Among the nuclear proteins the largest variation was in the histones. The only difference in the relative content of nuclear proteins between 7-day-old and adult brain was in the acidic deoxyribonucleoproteins which was higher in the younger animal. The electrophoretic profile of these proteins changed during brain development.     

人胚肝发育过程中染色质组份的变化

从14-,17-,21-,27-,34-,及38-周令的人胚肝细胞核分离出柒色质,分别对其中的RNA、DNA、组蛋白(HP)及非组蛋白(NHP)进行测定。在胚胎发育过程中肝柒色质HP/DNA比值变化不大。但是,NHP/DNA与NHP/HP比值发生显著改变。人胚肝NHP量的变化一直保持在整个胚胎发育过程中,NHP量的高峰位于21-及34-周。用SDS-聚丙烯酰胺凝胶板电泳分析处于不同发育阶段的人胚肝总染色质蛋白。电泳图谱显示出染色质NHP组分在质与量上有所改变。     

Specificity,avidity, and size of B-lymphocyte populations during ontogeny

The size and specificity of plaque-forming cell precursors (PFC) in murine fetal liver, neonatal, and adult spleen were studied in an adoptive transfer system. In this system, anti-4-hydroxy-3-iodo-5-nitrophenylacetic acid (anti-NIP) and anti-2,4,6-trinitrobenzene sulfonic acid (anti-TNP) direct PFC are generated from bone marrow-derived (B) cell precursors in fetal liver between 17 and 20 days of gestation and in 6- or 14-day neonatal spleen. PFC generated from fetal liver and neonatal and adult spleen cells are specific in that they lyse either NIP-coupled SRBC or TNP-coupled SRBC but not both. The generation of specific anti-NIP and anti-TNP PFC from precursors in fetal liver is primarily independent of antigenic stimulation. In contrast, the anti-NIP and anti-TNP responses generated from neonatal and adult spleen are antigen dependent. Both high-avidity PFC (detected with SRBC indicators coupled at low hapten density) and low-avidity PFC (detected with SRBC coupled at high hapten density) are generated from fetal liver and neonatal spleen cells; however, the proportion of high-avidity PFC precursors in adult spleen is at least threefold greater than in fetal liver or neonatal spleen. Analysis by velocity sedimentation indicates that most high-avidity PFC precursors are small lymphocytes in fetal liver, medium lymphocytes in 6-day neonatal spleen, and small lymphocytes in 14-day-old and adult spleen. Low-avidity PFC precursors are primarily medium-sized lymphocytes in fetal liver and 6-day neonatal spleen. In 14-day-old and adult spleen almost all high- and low-avidity PFC precursors are small lymphocytes. The results are discussed in terms of relative changes in the pool sizes of these lymphocyte populations.     

Analysis of the sinusoidal endothelial cell organization during the developmental stage of the fetal rat liver using a sinusoidal endothelial cell specific antibody, SE-1

The sinusoid organization during the development of fetal rat livers was studied using a SE-1 antibody, which we have previously established as a specific monoclonal antibody against rat sinusoidal endothelial cell (SEC). Expression and localization of the SE-1 antigen in the liver tissues of 13- to 21-day-old fetuses were immunofluorescently and immunoelectron microscopically examined. The first positive fluorescence was observed in the immature liver of 15-day-old fetuses. The initial positive staining was randomly distributed in the liver parenchyma and showed no direct relation to the large vessels which may be derived from the fetal vitelline veins. The positive linear staining increased in number and connected with each other during the course of development. The SE-1 staining pattern and the sinusoidal arrangement became similar to those of the adult liver after 20th day of gestation. Immunoelectron microscopically, the immature SEC showed a weak positive reaction for the SE-1 antigen at their membrane and was observed together with immature hepatocytes and hematopoietic cells in the 15-day-old fetal liver. Along with the liver development, SEC formed a sinusoid structure closely associated with hepatocytes and came to strongly express the SE-1 antigen. These results indicate that the organization of the hepatic sinusoid may start at around 15th day of the gestation and occurs randomly in the fetal liver parenchyma. It is also suggested that the expression of SE-1 antigen is possibly regulated by the intimate association with hepatocytes.     

Characterization of hepatic epidermal growth factor receptors in the developing rat

Binding of 125I-labeled epidermal growth factor (EGF) was characterized in basolateral plasma membranes prepared from the livers of 21-day gestation fetuses, 14-day-old sucklings and adult Sprague-Dawley rats using a self-generating Percoll gradient method. The membrane preparations employed have been previously assayed in terms of plasma membrane protein yield, enrichment of various marker enzymes and sodium-dependent bile acid and amino acid transport. 125I-EGF binding was saturable and time and temperature dependent. Equilibrium analyses showed that the suckling period is characterized by a marked decrease in overall hepatic EGF binding capacity (460 +/- 50 fmol/mg protein) compared to either the fetal period (1290 +/- 160 fmol/mg) or to adults of either sex (males = 1540 +/- 230, females 1010 +/- 130 fmol/mg). Treatment of the suckling rat with parenteral EGF resulted in a 78% reduction in the observed binding capacity when assessed 2 h after growth factor administration. Comparison of binding affinities revealed no significant difference between the suckling and adult preparations (Kd = 0.40 +/- 0.03 vs. 0.39 +/- 0.02 nM, respectively); however, both preparations differed significantly from the fetal group which exhibited a decreased affinity of binding with a higher overall dissociation constant (Kd = 0.68 +/- 0.06 nM). Thus, it appears that major ontogenetic changes occur in the rat hepatic ligand/receptor system for epidermal growth factor. These changes are discussed in the context of transitional events in mammalian development such as birth and weaning.     

Alteration in the regulation of plasma membrane glycoproteins of the hepatocyte during ontogeny

The expression of four integral membrane glycoproteins was examined in detail utilizing monospecific antibodies during liver development. These included asialoglycoprotein receptor, a hepatocyte glycoprotein residing in the sinusoidal domain, and three bile canalicular glycoproteins, leucine aminopeptidase, dipeptidyl peptidase IV, and a Mr 110,000 glycoprotein denoted GP 110. It was observed that asialoglycoprotein receptor, GP 110, and dipeptidyl peptidase IV were present in low amounts in fetal liver and reached adult levels between 1 to 3 weeks. In contrast, leucine aminopeptidase was present in nearly adult amounts in 18-day-old fetal livers. These observations were qualitatively confirmed by indirect immunofluorescent staining of frozen thin liver sections obtained from fetal and adult rats. Further, in fetal livers it was found that leucine aminopeptidase was not localized to typical bile canalicular areas. Immunoprecipitation studies performed in the presence of proteolytic inhibitors using detergent-solubilized extracts of metabolically labeled liver minces revealed that GP 110 was present in low amounts as Mr 110,000 and Mr 105,000 polypeptides in 17-day fetal livers but by 21 days of gestation the larger polypeptide was the major synthesis product. Conversely, the apparent molecular weights of leucine aminopeptidase and dipeptidyl peptidase IV were not altered during development. Experiments determining relative rates of synthesis using excess amounts of antibodies showed that the concentrations of the three bile canalicular glycoproteins in liver during ontogeny reflect their rates of synthesis. These results underscore that plasma membrane constituents of the hepatocyte undergo dramatic changes in expression and localization as the liver changes its physiological role at birth.     

Ontogeny of insulin-like growth factor I and insulin receptor kinase activity in rat liver.

IGF-I and insulin receptors possess tyrosine-kinase enzymatic activity considered to be essential for signal transduction and thereby mediating the putative effects of these hormones on fetal growth and development. We investigated the ontogeny of IGF-I and insulin receptor tyrosine-kinase activity in at least 3 separate membrane preparations from liver of rats at 21 day of embryonic life (21ED), 1 and 5 day of postnatal life (1PD and 5PD respectively) and adult. Receptors purified by wheat germ agglutinin chromatography (WGA) were exposed to graded concentrations of IGF-I or insulin, and tyrosine-kinase activity was measured by quantifying incorporation of 32P into the exogenous substrate poly[Glu,Tyr; 4:1]. IGF-I stimulated tyrosine-kinase solely at 1 PD as documented by a maximal increase of 346 +/- 167% over basal kinase activity with 6.6 nmol/L IGF-I. While the lack of response in adult animals could be explained by a striking decrease in receptors at that age, 125I-IGF-I binding and affinity labelling of the WGA preparations indicated substantial IGF-I receptors were present in the liver at each of the perinatal ages. Furthermore, this dissociation between IGF-I binding and the tyrosine-kinase activity of these IGF-I receptors could not be attributed to the presence/absence of IGF-I binding proteins as judged by affinity labelling. In contrast, insulin-stimulated tyrosine-kinase activity was observed at all ages tested although it appeared greatest at 1PD. We conclude that (i) expression of IGF-I tyrosine-kinase activity is linked to developmental events and differs from that found for the insulin receptor tyrosine-kinase activity, (ii) during the perinatal period there is an apparent dissociation between ligand binding by the IGF-I receptor and receptor tyrosine-kinase activity. These observations suggest modulation of IGF-I receptor tyrosine-kinase activity may be an important regulator of IGF-I action during the perinatal period.     

PROTEIN SYNTHESIS IN FRACTIONS FROM ISOLATED BRAIN CELL NUCLEI

Abstract— 1. The incorporation in vivo and in vitro of isotopically labelled leucine into fractions of nuclear proteins from young and adult rat brain was investigated.
2. During post-natal cerebral maturation, the ability of nuclei from brain cells to synthesize proteins decreased. The specific activities of all the fractions of nuclear protein were highest in 3-day-old rats and declined thereafter. Nuclei from adult brain cells exhibited only 10 per cent of the activity found in nuclei from brain cells of 3-day-old rats.
3. The 'residual protein' fraction was most rapidly labelled, peak activity being reached within 30 min after injection. In vitro , the 'residual protein' fraction attained maximum activity within 40 min.
4. The specific activity of the chromatin acidic proteins (HCl-insoluble) was considerably higher than that of the histones both in vivo and in vitro. Histones were the most inert of all the nuclear protein fractions studied.
The possible functional significance of the various protein fractions during the process of cerebral maturation and in the adult brain is discussed.     

Interactions of the nuclear thyroid hormone receptor with core histones

These studies concern the interactions of the rat liver thyroid hormone nuclear receptor with histones and factors influencing the receptor's assay and stability. Heating certain crude receptor preparations at 50 degrees C produces a selective loss of triiodothyronine (T3) but not thyroxine (T4) binding activity, whereas, with more purified preparations, such heating decreases both T3 and T4 binding. The selective T3-binding loss in crude preparations was found to be due to the simultaneous denaturation of the receptor's high-affinity hormone-binding activity for both T3 and T4 and generation of new low-affinity T4-binding sites. The fraction in which T4 binding can be activated could be separated from the receptors by Sephadex G-100 chromatography. Core histones stimulated both T3- and T4-binding activity of 6-fold-purified receptor preparations, and data from several different experimental approaches suggest that this stimulation is due to the capability of the core histones to prevent the receptor from binding to or being denatured by Sephadex G-25 assay columns. The core histones were also found to stabilize 500-fold-purified but not 6-fold-purified or crude receptor preparations. A number of other acidic or basic proteins had little or none of these stimulatory effects, whereas a few proteins (such as the insulin B chain and histone H1) did have activity, although it was less than that of the core histones. There were no significant differences between the purified core histone subfractions (H2A, H2B, H3, and H4). That core histones can interact with the thyroid hormone receptors was demonstrated more directly by the finding that the receptors bind to histone-Sepharose but not Sepharose or insulin- or ovalbumin-Sepharose columns and that this binding was blocked by core histones at concentrations suggestive of an affinity for the receptor-core histone interaction of around 3 microM at 0.15 M salt concentration. The results demonstrate the utility of the histones in the assay and stabilization of purified thyroid hormone receptors, but they fail to support our previous hypothesis of a receptor subunit where T3- but not T4-binding activity is regulated selectively by histones. However, the results indicate that histones may interact with the receptors with some degree of specificity, and they raise the possibility that the histones participate in the nuclear localization of the receptors.     

Different tissue distribution and hormonal regulation of messenger RNAs encoding rat insulin-like growth factor-binding proteins-1 and -2.

The insulin-like growth factor-binding proteins IGFBP-1 and IGFBP-2 are low mol wt IGFBPs that are similar in structure. They are not glycosylated and have a homologous amino acid sequence, including the number and position of 18 cysteine residues and a carboxyl-terminal Arg-Gly-Asp sequence that can be recognized by cell adhesion receptors. The present study demonstrates that expression of mRNAs encoding the two BPs differs in some fetal rat tissues and in the livers of adult rats after hypophysectomy, fasting, or streptozotocin-induced diabetes. As determined by Northern blot hybridization using cDNA probes for rat IGFBP-2 or human IGFBP-1, both mRNAs are expressed at high levels in liver of 21-day gestation and 1-day-old rats and at lower levels in 21- and 65-day-old rat liver. Levels of both mRNAs are higher in liver than in other fetal rat tissues. The relative abundance of the two mRNAs in most fetal tissues is similar to that in liver, except that kidney and brain have 8-fold and more than 25-fold higher relative levels of IGFBP-2 mRNA, respectively. IGFBP-2 mRNA is about 10- to 20-fold increased after hypophysectomy or fasting, whereas IGFBP-1 mRNA is relatively unchanged. IGFBP-2 mRNA levels are decreased completely by refeeding fasted rats for 3 days, but only partially decreased by treatment of hypophysectomized rats with GH, cortisone acetate, T4, and testosterone for 4 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fetal and postnatal development of cyclic GMP phosphodiesterase activity in the rat brain

Cyclic GMP concentration and cyclic GMP phosphodiesterase activity were studied in rat mothers and fetuses at 17, 19 and 21 days of intrauterine life and 0, 1, 4, 10, 15,20, 30 and 45 days after birth. During this developmental period, the increase in cyclic GMP concentration was discrete and the value in 15-day-old rats was already similar to the adult level. Cyclic GMP phosphodiesterase activity increased from 17- to 19-day fetuses and was significantly reduced in 21-day fetuses, neonates, and 1-day-old rats. This reduction may be a result of fetal endocrine preparation for parturition. During postnatal development, cyclic GMP phosphodiesterase activity increased in a parallel way in the limbic system, corpora striata, cerebral hemispheres, and diencephalon, reaching maximal level between 20 and 30 days after birth, and then decreasing to the adult value. The highest activity was found in corpora striata and the lowest in diencephalon. Cerebellar cyclic GMP phosphodiesterase activity was very high in the 4-day-old rat (257% of adult value) and diminished significantly in the 10-day-old rat with no subsequent changes. Kinetic analysis of the enzyme during postnatal forebrain development showed an increase in both the V_max and the apparent K_m. A decrease in the enzyme's V_max was observed only in the cerebellum.The importance of cyclic GMP phosphodiesterase regulation of cyclic GMP concentrations in the brain during development is discussed.     

Fractionation of rat-liver-chromatin nonhistone proteins into two groups with different metabolic rates.

In the pH interval 10.5-11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13. Dodecylsulfate-polyacrylamide gel electrophoresis revealed that the majority of the high-molecular-weight nonhistone proteins together with a few characteristic fractions with molecular weights of 40 000-60 000 remained in the alkali-resistant group. L-[14C]Leucine pulse-labelling experiments showed that the specific radioactivity of the alkali-labile nonhistone proteins was 2-3 times higher than that of the alkali-resistant nonhistone proteins, which, in turn, had the same specific radioactivity as that of the histones. The same held true for chromatin from regenerating rat liver. In the course of a 21-day chase the specific radioactivity of the alkali-labile nonhistone proteins gradually decreased and finally became 3 times lower than that of the alkali-resistant nonhistone proteins. On the contrary, the ratio of the specific radioactivities of the alkali-resistant nonhistone proteins and of the histones to the specific radioactivity of DNA remained constant during the chase. A conclusion can be drawn that a fraction of liver nonhistone proteins exists which is alkali-resistant and is conserved in chromatin like histones.     

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).     

Adenylate cyclase activity in crude plasma membranes of subcutaneous adipocytes during ontogenetic development of rats

In order to explain the differences in the hormone stimulated lipolysis during ontogenic development of rats, the activity of adenylate cyclase was determined in crude plasma membranes of subcutaneous adipocytes of 5, 14, 21 and 45 to 55-day-old animals. Stimulatory effects of nonhormonal and hormonal agents were expressed as the increment in percentage of basal values which were not significantly changed in the age groups studied. The highest stimulatory effect was observed after sodium fluoride in 14 and 21-day-old rats. Guanylylimidodiphosphate and GTP revealed the lowest stimulatory effects in adult animals (greater than 45-day-old). The beta-adrenergic agent isoproterenol revealed the highest stimulatory effect in the 5 and 45-day-old group while in the preparation from 14-day-old rats the adenylate cyclase activity was significantly lower. On the other hand, tetracosactide (beta 1-24-corticotropin) revealed the smallest stimulatory effect on the preparation from 5-day-old rats; its stimulatory effect steadily increased and reached the highest value in adenylate cyclase preparations from adult animals. It can be concluded that the adenylate cyclase system in subcutaneous adipocytes is already basically mature at early ontogenic stages of development in rats. Nevertheless, the explanation for the small variations of the enzyme activity in different age groups requires further study.     

Changes in subfraction composition of chicken lysine-rich histone during ontogenesis]

Lysine-rich histone isolated from different chicken tissues was separated electrophoretically into 4-5 subfractions. The subfrations reffered to as 1, 2, 3, and 4, occur in each the tissue studied, erythrocyte lysine-rich histone containing an additional subfraction 1a. F1 histone from mitotically active tissues (intestinal mucosa, thymus, testes) has a higher content of subfraction 2, while the same histones from mototically inactive tissues (liver, heart, brain) contain an elevated amount of subfraction 3. F1 histone isolated from liver, brain and heart of 21-day embryo has much more of subfraction 2, than the same histone of adult animal. During the chicken development from hatching till maturation the content of subfraction 2 in these organs decreases, and the content of subfraction 3 increases. The rate of this change in liver corresponds to the rate of DNA synthesis. In F1 histone of erythrocytes the content of subfraction 4 falls down during the post hatching ontogenesis.