Coordinated distribution patterns of three enzyme activities involved in the absorption and metabolism of beta-carotene and vitamin A along the villus-crypt axis of chick duodenum.

The conversion of beta-carotene to retinal and the succeeding metabolic process of the retinal leading to production of retinol and retinyl esters are the prerequisite for the utilization of beta-carotene as a provitamin A. These processes are participated by beta-carotene cleavage enzyme, retinal reductase and retinol esterifying enzyme(s) in the small intestine. To examine whether these enzymes exhibit the coordinated distribution in the villus, we have used the cryostat sectioning technique to quantify the activities of beta-carotene cleavage enzyme, retinal reductase and retinol esterifying enzymes along the villus-crypt axis in 8-day-old chick duodenum. The beta-carotene cleavage enzyme activity was very low in the crypt and gradually increased, reaching a maximum in the mid-villus. The villus-crypt gradient of the beta-carotene cleavage enzyme activity corresponded with those of retinal reductase activity and lecithin: retinol acyltransferase (LRAT) activity, but distinct from that of acyl-CoA: retinol acyltransferase (ARAT) activity. Furthermore, the distribution of the content of retinyl esters was similar to that of LRAT activity. These results suggest that the beta-carotene cleavage enzyme is coordinately distributed along the villus-crypt axis with retinal reductase and LRAT, the two enzymes which require cellular retinol-binding protein, typeII (CRBPII) as the donor of the substrate.     

Distribution of Ca2+-ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin D-dependent Ca2+-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca2+-ATPase activity and ATP-dependent Ca2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na+ + K+)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca2+-transport in basolateral membranes was highest in fraction II (8.2 +/- 0.3 nmol Ca2+/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca2+-transport activity (0.9 +/- 0.1 nmol Ca2+/min per mg protein). The distribution of high-affinity Ca2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca2+-transport. The activity of Na+/Ca2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca2+-transport system, the Na+/Ca2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.     

Effect of transforming growth factor-alpha on enterocyte apoptosis is correlated with EGF receptor expression along the villus-crypt axis during methotrexate-induced intestinal mucositis in a rat

The purpose of the present study was to evaluate the effect of transforming-growth factor-alpha (TGF-α) on enterocyte apoptosis following methotrexate (MTX) induced intestinal mucositis in a rat and in Caco-2 cells. Non-pretreated and pretreated with MTX Caco-2 cells were incubated with increasing concentrations of TGF-α. Cell apoptosis was determined by FACS cytometry. Adult rats were divided into four groups: Control, Control-TGF-α, MTX, and MTX- TGF-α rats. Three days later rats were sacrificed. Enterocyte apoptosis were measured at sacrifice. RT-PCR and Western Blotting was used to determine the level of Bax and Bcl-2 mRNA and protein. Real time PCR was used to measure epidermal growth factor receptor (EGFr) expression along the villus-crypt axis. The in vitro experiment has shown that treatment with TGF-α of Caco-2 cells results in a significant inhibition of cell apoptosis in a dose-dependent manner. In vivo experiment, a decreased levels of apoptosis in MTX- TGF-α rats corresponded with the decrease in Bax and with the increase in Bcl-2 at both mRNA and protein levels. The inhibiting effect of TGF-α on enterocyte apoptosis was strongly correlated with EGFr expression along the villus-crypt axis. In conclusion, treatment with TGF-α inhibits enterocyte apoptosis following MTX- injury in the rat.     

Immunoelectrophoretic studies on human small-intestinal brush-border proteins.

The amounts of lactase (beta-D-galactosidase, EC 3.2.1.23), sucrase (sucrose alpha-D-glucohydrolase, EC 3.2.1.48), maltase (alpha-D-glucosidase, EC 3.2.1.20) microvillus aminopeptidase (EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.-) in tangentially sectioned biopsies from jejunum were studied by quantitative immunoelectrophoresis and enzymic assays. All enzymes had their maximum activities near the mid-region of the villi and their lowest activities at the bases of the crypts. The ratio between enzyme activity and immunoreactive protein was constant along the villus-crypt axis. This result is consistent with a continuous brush-border-enzyme synthesis as the enterocytes migrate up the villi.     

Tentacle Morphogenesis in Hydra: A Morphological and Biochemical Analysis of the Effect of Actinomycin D

Stalked hydra exposed to 3 µg/ml actinomycin D for 24hr exhibited a restriction in the patterning of tentacle morphogenesis.The two-tentacied morphological modification manifested in regeneratinghydra was correlated with a reduction both in RNA synthesisin the hypostomal region of the animal and in the DMA syntheticactivity associated with normal tentacle elaboration. The tentaclesformed on actinomycin D-treated animals developed in the propersequence, however. In time the modification disappeared, indicatingthat the effect was reversible. Histological studies demonstrated depleted interstitial cell(I-cell) populations along the body column of actinomycin D-treatedhydra. Replacing the hypostomes of actinomycin D-treated hydrawith normal hypostomes reversed the cellular effects of actinomyinD exposure. All morphological and biochemical modifications in tentaclemorphogenesis occurring after actinomycin D treatment were consistentwith an impairment of hypostomal function in the animal. Evidencefrom ³H-actinomycin D autoradiography provided support for theproposal that the nervous system was the morphological siteof this malfunction.     

Effect of bacterial monoassociation on brush-border enzyme activities in ex-germ-free piglets: comparison of commensal and pathogenic Escherichia coli strains

This study was designed to investigate the effect of monoassociation of germ-free piglets with Escherichia coli strains on the development of intestinal brush-border enzyme activities. Piglets were delivered by hysterectomy, reared for seven days under germ-free conditions and fed milk formula diet. One group was maintained germ-free, the other four groups were monoassociated on day eight with one of four E. coli strains: non-pathogenic O86 or O83 and G58-1, or pathogenic 933D. The development of brush-border digestive enzyme functions in the small intestine was evaluated after 15 days. Germ-free controls exhibited slower developmental declines of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and delayed increases of sucrase and glucoamylase compared to conventionally grown animals. Association of germ-free piglets with the non-pathogenic E. coli strains O86 and O83 resulted in increased enterocyte differentiation along the length of the small intestine, accompanied by declining activities of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and elevated activities of maturational markers such as sucrase and glucoamylase. Maturational changes also occurred along the villus-crypt axis, as revealed by histochemical localization of aminopeptidase N on the villi tips in piglets colonized with E. coli O83. Interestingly, colonization with the pathogenic E. coli strain 933D stimulated changes in the main differentiation enzyme markers lactase, sucrase and glucoamylase to an extent comparable with those produced by the non-pathogenic and probiotic E. coli strains. In conclusion, germ-free piglets represent a valuable tool to study the consequences of colonization of the immature sterile gut with defined strains of bacteria.     

The effect of epidermal growth factor on the distribution of SGLT-1 in rabbit jejunum

The effect of epidermal growth factor (EGF) on the cellular and villous distribution of the sugar transporter SGLT-1 was examined. New Zealand White rabbits (1 kg) were anesthetized, and two jejunal blind loops were isolated and exposed to either 0.9% saline or EGF (60 ng/mL saline), for 1 h. In separate experiments, tissue was harvested for brush border membrane vesicles (BBMV), microsomal membranes, or fixed for immunohistochemistry. SGLT-1 was measured in membrane fractions by Western immunoblot or localized along the villus-crypt axis by immunofluorescent microscopy. EGF increased BBMV SGLT-1 content compared with paired controls. EGF stimulation also induced a corresponding decrease in microsomal SGLT-1 levels and induced the expression of additional SGLT-1 immunoreactivity further down the villus axis. The findings suggest that EGF upregulates intestinal glucose transport by stimulating the translocation of SGLT-1 from an internal microsomal pool into the brush border, thereby recruiting more villus enterocytes into the glucose transporting population.     

The effect of ethylene on RNA and protein synthesis in germinating Manketti (Ricinodendron rautanenii Schinz) seeds

Amino acid uptake and protein synthesis were monitored in the embryonic axis, cotyledons and endosperm of manketti seeds (Ricinodendron rautanenii Schinz) during imbition, dormancy and dormancy-breaking by ethylene. Protein synthesis increased in all tissues within 48 h of imbibition and was strongly inhibited by cycloheximide and actinomycin D. No marked increase in protein synthesis was observed following ethylene treatment suggesting that qualitative rather than quantitative changes in protein synthesis were associated with dormancy-breaking. Seed germination was relatively insensitive to treatment with actinomycin D in marked contrast to the inhibition observed with cycloheximide.It is suggested that dormancy-breaking by ethylene may require protein synthesis but not DNA-dependent RNA synthesis.     

Refractoriness to phosphaturic action of parathyroid hormone occurring independent of phosphate depletion in hyperparathyroid rats

In an attempt to clarify the underlying mechanism(s) in the disappearance of phosphaturic response to bolus parathyroid hormone (PTH) in hyperparathyroid patients, the effects of bolus bovine PTH (10 USP U) were studied in conscious thyroparathyroidectomized (T . PTX) male Wistar rats that had been infused with a dose of PTH (2.5 U/hr, for 16 hours) so as to reproduce hyperparathyroidism. These animals responded with an increase in urinary cyclic AMP, but without an increase in renal clearance of phosphate. The loss of phosphaturic response was not prevented by pretreatment with actinomycin D at a dosage close to full toxicity (0.1 mg/kg BW, ip, for 3 days). Actinomycin D at this dosage did not affect the normal stimulatory effects of bolus PTH on urinary cyclic AMP and renal clearance of phosphate in T . PTX rats. The continuous infusion of PTH produced nearly maximal phosphaturia throughout in the face of a significant depletion of phosphate. In addition, pretreatment with actinomycin D did not cause a further increase in urinary phosphate excretion during the infusion. These results, along with the report of Shah et al. (1979) indicating that the development of antiphosphaturic adaptation to acute phosphate depletion was prevented by comparable amounts of actinomycin D, indicate that the disappearance of phosphaturic response to bolus PTH by prior PTH infusion simply signifies the continuation of maximal phosphaturic response to the preceding PTH infusion. It is also suggested that the continuous action of PTH prevents, at least phenomenologically, the development of the gene-activation-mediated refractoriness to PTH or antiphosphaturia induced by acute phosphate depletion.     

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of administration of graded doses of oestradiol-17β and actinomycin D on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomes

1. The effects of graded doses of oestradiol-17beta and actinomycin D, administered separately or together, on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of uterine polyribosomes are described. Preparations of polyribosomes isolated from uteri of ovariectomized adult rats were determined for cytoplasmic concentration in vivo and assayed for [(14)C]leucine-incorporation activity in the cell-free system, exactly as described by Teng & Hamilton (1967b). 2. A minimal dose of 10mug of oestradiol-17beta administered for 10h was found to increase, by about 100%, both the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of the polyribosomes. A minimal dose of 250mug of actinomycin D administered for 10h was found to inhibit, by about 50%, the incorporation activity in vitro of the polyribosomes. All doses of the inhibitor administered for 10h failed to alter the cytoplasmic concentration in vivo of the polyribosomes. 3. A dose of 10mug of oestradiol-17beta restored to the control value the inhibitory effect of a dose of either 50 or 125mug of actinomycin D on the activity in vitro of the polyribosomes, at 10h after treatment with the inhibitor and the hormone. In these experiments, there was an increase of 60-100% in the cytoplasmic concentration in vivo of the polyribosomes. 4. A dose of 125mug of actinomycin D, administered to animals along with 10mug of oestradiol-17beta for 6-36h, abolished the hormone-induced enhancement of the incorporation activity in vitro, but did not prevent an increase of about 200% in the cytoplasmic concentration in vivo of the polyribosomes. However, treatment with 750mug of the inhibitor abolished both stimulatory effects of the hormone. 5. The results reported indicate that the stimulatory effects of oestradiol-17beta in vivo on the number and activity of the cytoplasmic polyribosomes in the uterus of the ovariectomized rat have different sensitivities to actinomycin D, but the primary molecular mechanisms responsible for the results are unknown. The major conclusion drawn is that the formation and appearance in the cytoplasm of newly formed polyribosomes are important features of the early action of oestrogen in the uterus.     

Distribution of Ca-ATPase, ATP-dependent Ca-transport, calmodulin and vitamin D-dependent Ca-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca²⁺-ATPase activity and ATP-dependent Ca²⁺-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na⁺ + K⁺)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca²⁺-transport in fraction II (8.2 ± 0.3 nmol Ca²⁺/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca²⁺-transport activity (0.9 ± 0.1 nmol Ca²⁺/min per mg protein). The distribution of high-affinity Ca²⁺-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca²⁺-transport. The activity of Na⁺/Ca²⁺ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca²⁺-transport system, the Na⁺/Ca²⁺ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.     

Tri-iodo thyronine regulates antioxidant enzyme activities in different cell fractions through a mechanism sensitive to actinomycin D in a teleost, Anabas testudineus (Bloch)

The present study evaluated the effects of hyperthyroid state on lipid peroxidation and antioxidant enzymes in the crude (CF), post nuclear (PNF) and mitochondrial fractions (MF) of the fish liver. The in vivo injection of T3 (200ng) did not change the lipid peroxidation products, malondialdehyde (MDA) and conjugated dienes (CD), while actinomycin D (10microg), a potent mRNA inhibitor when administered with T3 increased them. The antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) had an increased activity in CF and MF of hyperthyroid group to compete the increased oxidative stress, but actinomycin D partially inhibited the T3-induced activity. SOD and CAT activities in PNF of hyperthyroid group had no change, the glutathione concentration varied depending on the GPx and GR activity. Hyperthyroidism decreased the protein content, while simultaneous administration of actinomycin D inhibited the T3 action of elevating the protein content. The results suggest that the antioxidant defense status in A. testudineus is modulated by thyroid hormone, through an action sensitive to actinomycin D.     

Regulation of cytochrome P-450 dependent steroid hydroxylase activity in Manduca sexta: evidence for the involvement of a neuroendocrine-endocrine axis during larval-pupal development

Ecdysone 20-monooxygenase activity and the factors which may regulate this steroid hydroxylase were examined in the midgut of the tobacco hornworm, Manduca sexta, during the last larval stadium. Radioassay experiments revealed that midgut ecdysone 20-monooxygenase undergoes a single 50-fold increase in activity temporally coincident with the onset of the wandering stage. The increase in midgut monooxygenase activity was prevented by actinomycin D and cycloheximide, and could be elicited in head (but not thoracic) ligated animals by a brain-retrocerebral complex factor(s) released at the same time as prothoracicotropic hormone. In contrast, ecdysone or 20-hydroxyecdysone could elicit the increase in enzyme activity in both head and thoracic ligated animals. These data suggest the operation of a neuroendocrine-endocrine axis in the regulation of midgut ecdysone 20-monooxygenase activity.     

Comparison of the effects of human chorionic gonadotropin and luteininzing hormone on phosphofructokinase activity in isolated rat ovaries.

Phosphofructokinase activity in prepubertal rat ovaries is elevated by in vitro treatment with human chorionic gonadotropin or luteinizing hormone. The stimulatory effects of the two gonadotropins are additive. Puromycin and actinomycin D do not affect the enzyme increase induced by human chorionic gonadotropin, but the stimulatory action of luteinizing hormone is completely abolished by these antibiotics. These data suggest that the two hormones have different mechanisms of action and probably occupy different receptor sites on the ovarian cells.     

The interaction of catecholamines and adrenal corticosteroids in the induction of phosphopyruvate carboxylase in rat liver and adipose tissue

Catecholamines induced an increase in the activity of rat adipose tissue and liver phosphopyruvate carboxylases that was maintained for 48h. The response of adipose tissue phosphopyruvate carboxylase was blocked by actinomycin D, corticosteroids and propranolol, whereas corticosteroids and propranolol did not affect the liver enzyme. Cortisol phosphate, like actinomycin D, interfered only with the initiation of the increase in enzyme activity caused by noradrenaline, but not with the process of enzyme accumulation. In contrast, cycloheximide was effective in blocking enzyme induction throughout the course of the catecholamine effect. Adrenocorticotrophic hormone caused a short-term induction of adipose tissue phosphopyruvate carboxylase, which could be blocked by propranolol. Hepatic phosphopyruvate carboxylase, but not the adipose tissue enzyme, was induced by dibutyryladenosine 3':5'-cyclic monophosphate and by glucagon. Both nicotinic acid and nicotinamide decreased the normal induction of adipose tissue phosphopyruvate carboxylase caused by starvation, but only nicotinamide increased the activity of the liver enzyme.     

Insulin antagonises the growth hormone-mediated increase in the activity of phosphatidate phosphohydrolase in isolated rat hepatocytes

Rat hepatocytes were incubated in monolayer culture, under serum-free conditions for 8 h. Rat growth hormone (up to 100 nM) increased the activity of phosphatidate phosphohydrolase by up to 47%. Insulin (500 pM or 35 nM), cycloheximide or actinomycin D reversed this effect. The ability of growth hormone to modify the effects of insulin is discussed in relation to the control of the phosphohydrolase activity and glycerolipid synthesis.     

Oral and subcutaneous administration of cadmium chloride and the distribution of metallothionein and cadmium along the villus-crypt axis in rat jejunum

The route of Cd uptake influences the distribution of Cd, other metals, and metallothionein (MT). Although intestinal MT levels related to the tissue mass did not show proximodistal gradients after sc administration of CdCl₂, orally administered high doses of CdCl₂ increased mucosal MT levels longitudinally from the duodenum to the ileum. The gradient abolished when the mucosal MT level was related to the intestinal length. To further elucidate this finding, three groups of rats were studied: a control group, a group receiving dietary CdCl₂, and a group receiving sc injections of CdCl₂. The small intestine was removed after a 14-d treatment. Midjejunal segments were mounted in a cryomicrotome and cut transversally into five layers along the villus-crypt axis. Mucosal enzymes were measured to control these sections. Cd was measured by AAS and MT by RIA. Alkaline phosphatase and lactase activities exhibited the typical villus-crypt gradient. Mucosal MT levels paralleled those of Cd. Although Cd and MT concentrations were high at the tip of the villi and low in the crypts after oral administration, sc treatment reversed that profile. A molar Cd-MT ratio of approx 10 or 1 was reached after po or sc treatment, respectively. This demonstrates that only oral Cd may lead to an accumulation of Cd in the mucosal tissue fairly exceeding the binding capacity of small intestinal MT. The results show that different routes of Cd intake lead to a different MT-induction pattern in the intestinal wall and that longitudinal Cd and MT concentration gradients in the small intestine observed after high oral doses are a result of their high levels at the villus tips.     

Yolk deposition in Douglas-fir beetle oöcytes: Possible rôle of RNA synthesis in the follicular epithelium

RNA synthesis and morphological changes in the follicular epithelial cells of oöcytes of Douglas-fir beetle, Dendroctonus pseudotsugae were studied during the reproductive phase. Inhibition of synthesis of DNA dependent RNA by actinomycin D injections blocked yolk deposition in the oöcytes as well as oviposition within the normal period. A mixture of radioactively labelled haemolymph and ovarial proteins was deposited as yolk proteins in the oöcytes of normal beetles. Such proteins were not deposited in the oöcytes of females injected with actinomycin D; the blockage of yolk deposition persisted even when such females were treated extraneously with juvenile hormone.