Co-ordinated induction of beta-carotene cleavage enzyme and retinal reductase in the duodenum of the developing chicks

The developmental patterns of expression of beta-carotene cleavage enzyme activity were compared with those of retinal reductase and NAD-dependent retinol dehydrogenase activities in chick duodenum during the perinatal period. The beta-carotene cleavage enzyme activity was not detected in the duodenum before hatching, but it increased rapidly during 24 h after hatching. On the other hand, a considerable level of beta-carotene cleavage enzyme activity was observed in the liver of embryonic stages and its activity gradually rose during the perinatal period. Comparison of kinetic constants for the beta-carotene cleavage enzyme activities in the duodenum and the liver indicated that the enzyme in the duodenum possessed a lower affinity for beta-carotene than that in the liver. The retinal reductase activity was detected in the microsomes of the duodenum at the earliest time examined, i.e. day 16 of embryogenesis and its activity began to rise on the last day of embryogenesis, which was followed by a gradual increase until 1 day of age. The NAD-dependent retinol dehydrogenase activity was also seen in the microsomes of the duodenum in embryonic stages and its activity increased in parallel with the retinal reductase activity around the hatching period. These developmental inductions of beta-carotene cleavage enzyme and retinal reductase activities in the duodenum coincided with those of cellular retinol-binding protein, type II (CRBPII) and lecithin: retinol acyltransferase (LRAT). These results suggest that a co-ordinated induction mechanism should be operative for beta-carotene cleavage enzyme and retinal reductase, both of which are inevitable in the process of beta-carotene absorption and metabolism.     

Subcellular Localization and Developmental Changes of Aspartate-alpha-Ketoglutarate Transaminase Isozymes in the Cotyledons of Cucumber Seedlings

The total activity of aspartate-α-ketoglutarate transaminase in the cotyledons of cucumber (Cucumis sativus L.) seeds increased 17-fold during the first 2 days of germination in darkness and then declined gradually to 20% of the peak activity after 10 days. Exposure of the seedlings to light at day 3 accelerated the decline. The enzyme in the cotyledon extracts from seedlings at various ages was resolved into six distinct isozymes by starch gel electrophoresis. Isozymes 1 and 2 were glyoxysomal isozymes with different developmental patterns. Isozyme 1 followed the developmental pattern of the total enzyme activity in darkness, and was rapidly eliminated upon illumination. Isozyme 2 increased in activity to a peak at day 2 and declined rapidly thereafter, and disappeared completely at day 6; this developmental pattern was independent of light. No major difference in the optimal pH for activity, substrate specificity, and reversibility was observed between isozymes 1 and 2. The combined developmental pattern of isozymes 1 and 2 during germination correlated with that of the glyoxysomes. Isozyme 3 was located in the cytosol and its developmental pattern followed that of the total activity. Isozymes 4,5, and 6 were plastid isozymes and appeared only after 2 days of germination. Unlike many other chloroplast enzymes, the appearance of the chloroplast transaminase isozymes was under temporal control and was independent of illumination. No enzyme activity was detected in isolated mitochondria. The findings illustrate a complicated cellular control system for the appearance of various organelle-specific transaminase isozymes and thus the amino acid metabolism during germination.     

The development of mitochondrial oxidative enzymes in rat heart muscle.

Three different developmental patterns have been found in the heart muscle mitochondria: (a) Activity of inner membrane enzymes, succinate-cytochrome c reductase and rotenone-sensitive NADH-cytochrome c reductase, was found to increase rapidly after birth till the 25th day; no further increase was found till the 60th day. Both brances of the respiratory chain, i.e. NADH-dependent and flavoprotein-linked were found to develop in parallel. (b) Activity of retoenone insensitive-NADH cytochrome c reductase, an outer membrane enzyme, did not show any change during developement. (c) Activity of monoamine oxidase, another outer membrane enzyme, was found to increase after the 10th day of postnatal life and the increase in activity continued till the 60th day.     

Fructose-1,6-diphosphatase activity in brown adipose tissue of the developing rat.

An enzyme activity capable of converting fructose-1,6-diphosphate to fructose-6-phosphate was demonstrated to present in crude tissue extracts from brown adipose tissue of the rat. Mg2+ was essential for the expression of activity. EDTA (0.5 mM) increased the activity by 30%. Fructose-1,6-diphosphate in concentrations of 1 and 10 mM inhibits activity by 30% and 60% respectively. A 65% inhibition was observed in the presence of 0.2 micrometer 5' AMP. The activity of the enzyme was measured in rat brown adipose tissue at different stages of development. It rises sharply between day 2 and day 6 and continues to increase reaching a maximum between 6 and 11 days. Thereafter the activity gradually declines to values observed prenatally. The normal developmental rise in activity could be prevented by chemical sympathectomy on day 2. This procedure had no effect when carried out on day 9. There was a significant increase in enzyme activity after cold adaptation. The possible physiological significance of this enzyme in brown adipose tissue is discussed.     

Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein

Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP:phosphatidate cytidylyltransferase and CDPdiacylglycerol:inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7-10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP:phosphatidate cytidylyltransferase in rat brain is reported here for the first time.     

Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein

Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP: phosphatidate cytidylyltransferase and CDPdiacylglycerol: inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7–10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP: phosphatidate cytidylyltransferase in rat brain is reported here for the first time.     

Development of glycogen and phospholipid metabolism in fetal and newborn rat lung.

Glucose, a major metabolic substrate for the mammalian fetus, probably makes significant contributions to surface active phospholipid synthesis in adult lung. We examined the developmental patterns of glycogen content, glycogen synthase activity, glycogen phosphorylase activity and glucose oxidation in fetal and newborn rat lung. These patterns were correlated with the development of phosphatidylcholine synthesis, content and the activities of enzymes involved in phosphatidylcholine synthesis. Fetal lung glycogen concentration increased until day 20 of gestation (term is 22 days) after which it declined to low levels. Activity of both glycogen synthase I and total glycogen synthase (I + D) in fetal lung increased late in gestation. Increased lung glycogen concentration preceded changes in enzyme activity. Glycogen phosphorylase a and total glycogen phosphorylase (a + b) activity in fetal lung increased during the period of prenatal glycogen depletion. The activity of the pentose phosphate pathway, as measured by the ratio of CO2 derived from oxidation of C1 and C6 of glucose, declined after birth. Fetal lung total phospholipid, phosphatidycholine and disaturated phosphatidylcholine content increased by 60, 90 and 180%, respectively, between day 19 of gestation and the first postnatal day. Incorporation of choline into phosphatidylcholine and disaturated phosphatidylcholine increased 10-fold during this time. No changes in phosphatidylcholine enzyme activities were noted during gestation, but both choline phosphate cytidylyltransferase and phosphatidate phosphatase activity increased after birth. The possible contributions of carbohydrate derived from fetal lung glycogen to phospholipid synthesis are discussed.     

Unusual electrophoretic patterns for phosphoglucomutase and fumarase in a population of Lecithochirium rufoviride (Trematoda: Hemiuridae), a parasite of Conger conger.

Electrophoretic analyses of phosphoglucomutase (PGM) and fumarase (FH) in a population of Lecithochirium rufoviride parasitizing Conger conger, revealed 2 independent activity zones for each enzyme on starch gel electrophoresis. However, some individuals exhibited only 1 activity zone for 1 or both enzymes. The banding patterns observed strongly suggest that (1) PGM is coded by 2 polymorphic loci, Pgm-1 (expressed in all individuals) with allelic frequencies not significantly different from those expected under Hardy-Weinberg equilibrium, and Pgm-2 (expressed in a subset of individuals); and (2) FH is coded by 2 loci, Fh-2 (monomorphic and expressed in all individuals) and Fh-1 (expressed in a subset of individuals). A high degree of concordance (88.75%) was observed between the expression and nonexpression of Pgm-2 and Fh-1. The most likely explanations for these findings are either variation in enzyme expression with developmental stage or the presence of null alleles at high frequencies in the population.     

Immunogold localization of nodule-enhanced phosphoenolpyruvate carboxylase in alfalfa

Phosphoenolpyruvate carboxylase (PEPC; EC 4-1-1-31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N₂-fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′-upstream region of the PEPC gene fused to β-glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N₂-fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.     

Enzymes of purine metabolism in the nuchal muscle (M. complexus) of chick embryo]

The developmental patterns of enzyme activities related to GMP metabolism have been investigated in chick embryo musculus complexus (m. Complexus). Guanylate phosphatase activity increases conspicuously from 18th to 21st day, guanosine phosphorylase increases on the 21st day and the guanase shows a very low activity during the whole period considered. Xanthine oxidase was always found absent. The results suggest that during the first period of incubation GMP breakdown in chick embryo m. complexus might follow a catabolic pathway, while starting from the 18th day some guanine might be converted to GMP originating a new metabolic pathway as previously suggested for AMP metabolism.     

Comparison of the developmental patterns of mitochondrial malate dehydrogenase between mung bean and cucumber cotyledons during and following germination

The development of mitochondrial NAD⁺-malate dehydrogenase (EC 1.1.1.37) in mung bean and cucumber cotyledons was followed. using the antibody raised against it, during and following germination. The developmental patterns were quite different between the two. In cucumber, the content of mitochondrial malate dehydrogenase continued to increase through 3–4 days after the beginning of imbibition. This was, at least in part, due to active synthesis of the enzyme protein, and the synthesis seemed to be regulated by the availability of the translatable mRNA for the enzyme. In mung bean, on the other hand, the enzyme was present in dry cotyledons at a rather high concentration, and remained at a constant level between day 1 and day 3 after the reduction of the content to one-half its initial level during the first day. De novo synthesis of the enzyme could not be detected in mung bean cotyledons by pulse-labeling experiment.     

Endogenous peroxidase in the visceral endoderm of early mouse embryos

Endogenous peroxidase activity was demonstrated in early mouse embryos by means of the diaminobenzidine staining reaction. This enzyme was observed in visceral endoderm on the seventh to eighth day of gestation in vivo, but was no longer detected on the ninth day of development. In cell layers developing from blastocysts or isolated inner cell masses cultured for 96-144 h (developmental stage equivalent to 6-7.5-day-old embryos), diaminobenzidine product was also observed in visceral endodermal cells. Most of the endogenous peroxidase was localized inside or close to the numerous apical vacuoles in the endoderm. Ectoderm, mesoderm, ectoplacental cone, and trophoblast cells did not contain endogenous peroxidase.     

THE DEVELOPMENT OF D-AMINO ACID OXIDASE IN RAT CEREBELLUM

D-Amino acid oxidase (D-amino acid: O₂ oxidoreductase (deaminating), EC 1.4.3.3; D-AAO) activity is biochemically undetected in rat brain stem, cerebellum and forebrain until 14 days after birth. Adult levels are attained by day 30 in the brain stem, and by day 36 in the cerebellum. At adulthood, forebrain D-AAO activity per g wet weight of tissue is less than 2% that of the cerebellum. In contrast to the pattern in the CNS, substantial D-AAO activity is present in both liver and kidney 2 days before birth and adult levels are approached within 2 weeks of birth. Nonetheless, D-AAO activities in rat liver, kidney, brain stem and cerebellum are likely to be due to a single enzyme which has properties very similar to the purified hog D-AAO. The late ontogenesis of D-AAO activity in cerebellum and brain stem relative to that in liver and kidney parallels reported phylogenetic data. Histochemical staining for D-AAO in rat cerebellar cortex is absent until 15 days after birth when activity is first observed in some cells of the external germinal zone and adjacent molecular layer. These cells appear to migrate to a final destination around the Purkinje cell soma and leave processes at the pial surface. By 21 days of age an adult pattern of staining is manifest throughout the cerebellum but it is of weak intensity. The adult pattern includes some staining in the granular layer which seems to be associated with mossy fibers and certain cerebellar glomeruli, and strong staining at the pial surface, in the molecular layer, and in cells surrounding, but not within, the Purkinje cell soma. The data suggest that the biochemical appearance of D-AAO in developing cerebellum derives from two sources: one associated with differentiation of one of the last cell types to form from the external germinal zone, and the other with maturation of mossy fibers and their synapses (cerebellar glomeruli).     

Studies on sex-organ development. Effect of hormones on ornithine decarboxylase activity in the developing chick ovary

The response of ornithine decarboxylase activity to hormones in the embryonic left ovary was measured throughout the stages of development. During the early stage of ovarian development (9th day of incubation), the ornithine decarboxylase activity (in terms of pmol CO₂/30min per mg of protein) was high (766); it decreased from the 10th to the 12th day (575–239), increased slightly from the 13th to the 15th day (306) and finally fell to a low value (192–20) from the 18th day of development to birth. Administration of an optimal dose of oestrogen to the 9–10-day embryo stimulated the ovarian ornithine decarboxylase activity by 48–53%. If the same dose of oestrogen was administered to the 15–18-day embryo, the ovarian enzyme activity was suppressed by 32–43%. This biphasic response to oestrogen for enzyme induction is characteristic of the developing ovary and is not observed in other genital organs of the chick. In the early developmental stage (9–10th day) testosterone has no effect on ovarian ornithine decarboxylase activity, but in the late stage testosterone inhibits the activity by 41%. Organ culture techniques have been used to test the ovarian response to lutropin (luteinizing hormone). Lutropin stimulated ornithine decarboxylase activity by approx. 99–155% in the ovary of the early embryonic stage (10–13th day), and by 175–200% in the ovary of the late embryonic stage (15–18th day). The alteration in enzyme activity in the ovary as assayed in vitro during development is not due to the effect of the size of the endogenous ornithine pool. The relationship of ornithine decarboxylase activity to the morphological and biochemical changes in the developing ovary is discussed.     

Ontogenetic changes in hepatic glutathione system (synthesis, catabolism, export) of male Uje:WIST rats.

The age-courses of concentrations of reduced (GSH) and oxidized (GSSG) glutathione, of GSH synthesizing enzyme activities, of glutathione S-transferase (GST), of GSSG-reductase (GR) and of biliary GSH and GSSG export were measured in livers from male Uje:WIST rats. Additionally, the age-courses of plasma GSH and GSSG concentrations were investigated. The hepatic level of GSH showed a biphasic pattern with a first maximum immediately after birth and a small second peak at the 50th day of life. The GSSG level increased continuously up to day 60 of life. The cytosolic GSH synthesizing enzyme activities showed diverse developmental patterns indicating different regulation principles. The hepatic activity of GR was relatively constant in the different age groups after birth. The GST activity (with o-dinitrobenzene as substrate) was relatively low at birth (about 30% of the maximum measured at day 60 of life). The maximum of GSH plasma level was found at birth. With increasing age a significant decrease in this level was observed. The excretion rate of total GSH (GSH + 2 GSSG) in bile was found to increase about 9-fold between 15 and 105 days of age. The results indicate that changes of hepatic GSH concentration with age are dependent on numerous factors. The balance between synthesis, catabolism and export is important for the maintenance of this level.     

Chondrocyte hypertrophy can be induced by a cryptic sequence of type II collagen and is accompanied by the induction of MMP-13 and collagenase activity: implications for development and arthritis.

The objective of this study was to determine whether a peptide of type II collagen which can induce collagenase activity can also induce chondrocyte terminal differentiation (hypertrophy) in articulate cartilage. Full depth explants of normal adult bovine articular cartilage were cultured with or without a 24 mer synthetic peptide of type II collagen (residues 195-218) (CB12-II). Peptide CB12-II lacks any RGD sequence and is derived from the CB12 fragment of type II collagen. Type II collagen cleavage by collagenase was measured by ELISA in cartilage and medium. Real-time RT-PCR was used to analyze gene expression of the chondrocyte hypertrophy markers COL10A1 and MMP-13. Immunostaining for anti-Ki67, anti-PCNA, (proliferation markers), type X collagen, cleavage of type II collagen by collagenases (hypertrophy markers) and TUNEL staining (hypertrophy and apoptosis markers) were used to detect progressive maturational stages of chondrocyte hypertrophy. At high but naturally occurring concentrations (10 microM and up) the collagen peptide CB12-II induced an increase in the expression of MMP-13 (24 h) and cleavage of type II collagen by collagenase in the mid zone (day 4) and also in the superficial zone (day 6). Furthermore the peptide induced an increase in proliferation on day 1 in the mid and deep zones extending to the superficial zone by day 4. There was also upregulation of COL10A1 expression at day 4 and of type X staining in the mid zone extending to the superficial zone by day 6. Apoptotic cell death was increased by day 4 in the lower deep zone and also in the superficial zone at day 7. The increase in apoptosis in the deep zone was also seen in controls. Our results show that the induction of collagenase activity by a cryptic peptide sequence of type II collagen, is accompanied by chondrocyte hypertrophy and associated with cellular and matrix changes. This induction occurs in the mid and superficial zones of previously healthy articular cartilage. This response of the chondrocyte to a cryptic sequence of denatured type II collagen may play a role in naturally occurring hypertrophy in endochondral ossification and in the development of cartilage pathology in osteoarthritis.     

Developmental changes affected by Mn deficiency

The changes in tissue Mn, Cu, Fe, Zn, and Superoxide dismutase (SOD) activity were studied in control and Mn-deficient mice during postnatal development. Mn levels were lower in tissues from Mn-deficient mice than in controls throughout development. By day 60, Mn concentration in tissues from Mn-deficient mice was at least 70% lower than that of controls. Cu levels in the two groups did not differ appreciably. Liver Cu concentration was highest at day 5, then decreased. Heart and kidney Cu increased throughout development. Fe concentration in heart and liver was similar in both groups at 1, 5, and 20 days of age, but at day 60, kidney Fe in the Mn-deficient mice was 40% higher than in controls. The developmental pattern for MnSOD activity paralleled that of Mn concentration. At day 5, there were no differences in MnSOD activity between control and deficient mice. By day 60, MnSOD activity in most tissues was at least 50% lower than that of controls, possibly increasing the susceptibility of the Mn deficient animal to oxidative damage. These developmental patterns should help investigators to determine the tissues and time periods in which to study trace element metabolism.     

The effect of continuous irradiation on cell proliferation and maturation in small intestinal epithelium.

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with 3H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36-48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.     

Giardia lamblia: expression of alkaline phosphatase activity in infected rat intestine

Alkaline phosphatase (IAP) is a marker of intestinal microvillus membrane. Changes in IAP activity have been studied as a function of Giardia lamblia (G. lamblia) infection using rat as the experimental model. At day 11 and 15 post-infection, enzyme activity was reduced (p<0.01) compared to controls. The enzyme levels were essentially similar to control values by day 30 post-infection. Analysis of the enzyme activity in cell fractions across crypt-villus axis revealed a marked decrease in enzyme activity in the villus tip and mid villus regions but a considerable increase (p<0.01) in enzyme activity in the crypt base of 11 day post-infected animals compared to that in controls. The observed changes in IAP activity were confirmed by assaying the enzyme activity in acrylamide gels using bromo-chloro-indolyl phosphate staining under non-denaturing conditions. These findings indicate differential changes across the crypt-villus axis, but impaired alkaline phosphatase levels in G. lamblia infected rat intestine.     

THE EFFECT OF CONTINUOUS IRRADIATION ON CELL PROLIFERATION AND MATURATION IN SMALL INTESTINAL EPITHELIUM

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with ³H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36–48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.