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A M Lennon

Publications and source records attributed to A M Lennon.

At least 37 records · Page 2Linked to original sources
PubMed1997

Isolation of a B-cell-specific promoter for the human class II transactivator.

The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex (MHC) class II antigens. The tissular patterns of CIITA and MHC class II gene expression are tightly correlated: CIITA mRNA is highly expressed in B cells, and is induced by interferon gamma (IFN-gamma) in macrophage and epithelial cell lines. We first isolated two overlapping cosmids encoding human CIITA which, when co-transfected, are able to restore MHC class II expression in a B-lymphoblastoid cell line (B-LCL) defective for CIITA. Subsequently, a 1.8 kilobase (kb) fragment of the CIITA promoter was isolated and sequenced. A motif presenting a strong similarity to an initiator was detected, as well as putative binding sites for Sp1, GATA-2, LyF-1, ets-1, AP1, and MZF1 transcription factors, and two GAS motifs. When introduced in front of a luciferase reporter gene, this promoter is able to direct a high luciferase activity in a human B-LCL. In contrast, luciferase expression was not stimulated after IFN-gamma treatment when the construct was transfected in macrophage or in epithelial cell lines. However, an induction of the human CIITA gene was observed in mouse macrophage and fibrosarcoma cell lines, when the cells were transfected with a cosmid containing the human CIITA gene, but lacking the 1.8 kb promoter described above. Taken together, these data suggest the existence of an intragenic promoter driving an IFN-gamma-inducible expression of CIITA.

B-Lymphocytes↗
PubMed1994

Induction of type III-deiodinase activity in astroglial cells by retinoids.

Thyroid hormones and retinoic acid (RA) are important modulators of growth, development, and differentiation. Type III deiodinase (D-III), which catalyzes thyroid hormones degradation in the brain and in cultured astroglial cells, is induced in astroglial cells by multiple pathways, including cAMP, 12.0-tetradecanoylphorbol-13-acetate (TPA), fibroblast growth factors, and thyroid hormones themselves. In the present study, the effects of retinoids on D-III activity were examined in astroglial cells cultures in a chemically defined medium devoid of hormones and growth factors. Incubation of astroglial cells with 5 microM all-trans-RA caused up to 200-fold increase in D-III activity, which reached a plateau after 48 h. The retinoid-induced increase in D-III activity was concentration dependent (0.5 microM all-trans-RA and 9-cis-RA producing half-maximal effect). Retinol was effective at physiological concentrations (1 and 10 microM). The 48 h effects of 5 microM all-trans-RA and 10 nM thyroid hormones on D-III activity were at least additive. Addition of 2 nM acidic fibroblast growth factor or 1 mM 8-bromo-cAMP for the last 8 h of a 48 h incubation with 5 microM all-trans-RA did not alter the induction by all-trans-RA, whereas 0.1 microM TPA in the same conditions produced an additive effect with all-trans-RA. All-trans-RA (5 microM) had little or no effect on type II deiodinase, the enzyme which catalyzes the activation of thyroxine to 3,5,3'-triiodothyronine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

12-O-tetradecanoylphorbol 13-acetate and fibroblast growth factor increase the 30-kDa substrate binding subunit of type II deiodinase in astrocytes.

Type II 5'-deiodinase (D-II) catalyzes the intracellular conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) in the brain. The D-II activity in astroglial cell cultures is induced by several pathways including cyclic AMP (cAMP), 12-O-tetradecanoylphorbol 13-acetate (TPA), and fibroblast growth factors (FGFs). We have examined the effect of TPA and FGFs on the 30-kDa substrate binding subunit of D-II, by affinity labeling with N-bromoacetyl-[125I]T4 in astroglial cells. TPA (0.1 microM), 20 ng/ml acidic FGF (aFGF), and 1 mM 8-bromo cyclic AMP all caused an increase in the 30-kDa protein. cAMP induced the greatest increase (fivefold) followed by TPA (3.2-fold) and FGF (2.8-fold). Glucocorticoids acted synergistically with cAMP and aFGF and promoted the effect of TPA. Affinity labeling was competitively inhibited by bromoacetyl-T4 > bromoacetyl-T3 > T4 > reverse T3 > iopanoic acid > T3 > 3,5,3'-triiodothyroacetic acid. The effect of TPA (0.1 microM) was maximum at 8 h and then gradually decreased. aFGF (20 ng/ml) plus heparin (17 micrograms/ml) induced a maximal 30-kDa increase at 8 h, which stayed stable for up to 24 h. The effect of aFGF was concentration dependent. Of the other growth factors studied, only basic FGF and platelet-derived growth factor induced small increases in the 30-kDa protein. Epidermal growth factor had little effect. In vitro labeling of cAMP, TPA, and aFGF-stimulated cell sonicates resulted in an increase in the 30-kDa protein that paralleled the increase in D-II activity.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Sulfation after deiodination of 3,5,3'-triiodothyronine in rat cultured astrocytes.

The metabolism of [125I]T3 by rat astrocytes in culture was analyzed by Sephadex LH-20 chromatography and HPLC. The conjugates isolated on LH-20 were not hydrolyzed by glucuronidase, indicating the absence of glucuroconjugates. 3,3'-Diiodothyronine (3,3'T2) sulfate (3,3'T2-S) was the main product that accumulated in the medium over the T3 concentration explored (10 pM to 10 nM). The identity of the peak eluted as 3,3'T2-S was ascertained by its hydrolysis with sulfatase and the generation of 3,3'T2 identified by HPLC. 3'-Monoiodothyronine sulfate was also found in cells treated with 1 microM retinoic acid, i.e. with high type III deiodinase activity. No T3 sulfate (T3-S) was found as a metabolite of T3. Astrocytes did not break down 1 nM [125I]T3-S added to the medium. Astrocytes pretreated for 3 days with 10 nM T3 showed increased production of 3,3'T2-S from 10 nM [125I]T3. Exogenous [125I]3,3'T2 (20 nM) was conjugated to 3,3'T2-S released into the medium. Pretreatment of astrocytes with 10 nM T3 did not alter the production of 3,3'T2-S from 3,3'T2. Thus, T3 is metabolized in astrocytes by direct 5-deiodination, followed by sulfation. Whereas T3 induces its own deiodination and type III deiodinase activity, T3 does not regulate the sulfation of its main metabolite, 3,3'T2. This demonstration of sulfation of iodothyronines in cells originating from the brain raises the question of the role of this TH metabolic pathway in the brain.

Animals↗
PubMed1992

Purification and characterization of rat brain cytosolic 3,5,3'-triiodo-L-thyronine-binding protein. Evidence for binding activity dependent on NADPH, NADP and thioredoxin.

A rat brain cytosolic 3,5,3'-triiodo-L-thyronine-(T3)-binding protein (CTBP) was purified using, successively, carboxymethyl-Sephadex, DEAE-Spherodex, T3-Sepharose-4B affinity chromatography and Sephacryl S-200. The molecular mass determined by SDS/PAGE wa 58 kDa. The binding characteristics determined by Scatchard analysis revealed a single class of binding sites with a Ka of 1.56 nM-1 and a maximal binding capacity of 7500 nmol T3/g protein. The relative binding affinities of iodothyronine analogues were D-T3 > L-T3 > L-T4 > 3,3'-5-triiodothyroacetic acid > reverse T3. The optimum pH for binding was 7.5. Purified brain CTBP was reversibly inactivated by charcoal. NADPH, NADP and thioredoxin restored binding activity to a level higher than that of the control; this effect was concentration dependent. Maximal activation was observed at 25 nM NADPH. NADP was effective only in the presence of 1 mM dithiothreitol; maximal activity was obtained at 10 nM NADP. At concentrations higher than 50 nM NADP, the binding gradually decreased. Thioredoxin in the presence of 1 mM dithiothreitol activated CTBP; maximal binding was obtained with 4 microM thioredoxin. In the presence of NADPH, NADP or thioredoxin the maximal binding capacity increased 2-4 times and the Ka was 2.6 nM-1. These results show that the activity of purified cytosolic brain T3-binding protein may be modulated by NADPH, NADP or thioredoxin.

Animals↗
PubMed1992

Induction of type III deiodinase activity in astroglial cells by thyroid hormones.

The type III deiodinase (D-III) activity in astroglial cells is induced by multiple pathways activated by cAMP, 12-O-tetradecanoylphorbol-13-acetate (TPA), and fibroblast growth factors (FGFs). This study examines the effects of thyroid hormones on D-III activity in astroglial cells with or without induction by these factors. Addition of 10 nM T3 to the culture medium caused a slow increase in D-III activity, which reached a plateau after 48 h. This increase was concentration dependent (maximal response at 10 nM). Doses as low as 0.3 nM caused significant increases in D-III activity. The effect of T3 was reversible. A dose of 10 nM L-T3, D-T3, T4, 3,5,3'-triiodothyroacetic, or 3'-isopropyl-3,5-diiodothyronine produced 5- to 15-fold increases in D-III activity after 48 h. In contrast, 10 nM L-thyronine, 3-monoiodothyronine, 3,3'-diiodothyronine, 3,5-diiodothyronine, and rT3 were without effect. A dose of 10 nM T3 or T4 amplified the D-III activity stimulated by 0.1 microM TPA, 20 ng/ml acidic FGF, or 1 mM 8-bromo-cAMP 3- to 8-fold. Otherwise, T3 rapidly inhibited D-II activity. This inhibition was concentration dependent, with a half-maximal effect around 10 nM. In conclusion, thyroid hormones induce D-III activity and potentiate the D-III activity induced by cAMP, TPA, and FGFs in astroglial cells. These reversible effects together with inhibition of D-II activity may contribute to protect the brain against hyperthyroidism.

Animals↗
PubMed1988

Characterization of a cytosolic triiodothyronine binding protein in atrium and ventricle of rat heart with different sensitivity toward thyroid hormone levels.

Cytosolic T3-binding protein (CTBP) has been identified in both the ventricle and atrium of adult rat hearts. Its biochemical characteristics and concentration have been determined in the two tissues as a function of thyroid hormone level. In both tissues association and dissociation constants were, respectively, k+1 = 1.3 x 10(8) M-1/min and k-1 = 0.025 min-1. Scatchard analysis of T3 equilibrium binding data revealed a single class of binding sites (Ka = 3.8 x 10(8) M-1). The maximal binding capacity (MBC) was 1400 fmol/mg protein in the ventricle and 730 fmol/mg protein in the atrium. The apparent mol wt of CTBP, determined by gel filtration, was 63.000. Among the thyroid hormone analogs tested in ventricular cytosol, D-T3 had the highest affinity, followed by L-T3, L-T4, 3,3',5-triiodothyroacetic acid, and rT3. These characteristics were very similar to those previously described for rat brain, and dog and rat liver and kidney CTBP. In hypothyroid rats MBC was only increased in the atrium (50-100%); after a single injection of T4 (2 micrograms/10 g BW 3 or 18 h before death) values returned to normal in the atrium and declined in the ventricle (-35%). During postnatal development, the highest MBC value (2000 fmol T3/mg protein) was observed in atria on day 10, i.e. when the serum T4 level was still low, and in the ventricle on day 30 (4000 fmol T3/mg protein) when the serum T4 level was at its highest. Binding affinities were similar in the two tissues at all ages studied. It was twice as high in both these tissues during the first week of development than in adulthood. These results favor a thyroid hormone down-regulation of the binding capacity of CTBP that would be more sensitive to the hormone in the atrium than in the ventricle.

Aging↗
PubMed1985

Cellular location of cytosolic triiodothyronine binding protein in primary cultures of fetal rat brain.

The evolution of a cytosolic triiodothyronine (T3) binding protein was studied in primary cultures of fetal rat brain. These cultures exhibited neuronal characteristics during the first week. T3 binding activity in cell supernatants increased during this period from 39 +/- 7 (mean +/- SD) to 159 +/- 24 fmoles T3/culture flask. A similar increase was observed in the soluble proteins. After day 8, neuronal death occurred and glial cells multiplied and differentiated. On day 11 an 86% drop in the binding activity was observed (24 +/- 7 fmoles T3/culture flask); the pool of soluble proteins remained stable. Scatchard analysis revealed two types of binding site in both 7- and 14-day cultured cell cytosols. Binding affinities were similar in both cytosols (KA1 approximately 1.5 X 10(9) M-1, KA2 approximately 1 X 10(8) M-1); in contrast, the number of sites was 4-fold smaller in 14-day cytosols. In subcultures mostly composed of glial cells, almost the same affinities were measured, but the numbers of both types of sites were 20 times smaller than in 7-day cells. These results show that in cell cultures from embryonic rat telencephalon, cytosolic T3 binding protein is mainly located in the neurons.

Animals↗
PubMed1983

A high affinity thyroid hormone binding protein in the cytosol of embryonic rat brain cells in primary cultures.

A thyroid hormone binding protein(s) has been characterized in the cytosol of fetal rat brain cells in primary cultures. This protein is closely related to the one described in brain supernatants with respect to its electrophoretic mobility, binding kinetic parameters and estimated molecular weight (65 000 daltons). However, in contrast to the brain cytosolic binding protein, two classes of affinity sites for triiodothyronine (T3) and thyroxine (T4) have been demonstrated: a high affinity site (KA = 1.2-3.7(3) X 10(9) M-1 for T3 and KA = 3.7-5 X 10(8) M-1 for T4) and a low affinity site (KA = 0.8-1.4 X 10(8) M-1 for T3 and 1.6-2.9 X 10(7) M-1 for T4). The results are discussed with respect to their cellular significance.

Animals↗
PubMed1982

Heterogeneity of microtubule-associated proteins and brain development.

Developmental changes in the composition of brain microtubule-associated proteins have been studied in three species: the rat and the mouse, which are characterized by post-natal brain development, and the guinea-pig, whose brain is mature at birth. 1. At an adult stage, and whatever the species, two major microtubule-associated proteins, which have been referred to MAP2 and tau, have been identified by polyacrylamide gel electrophoresis. Rat tau is composed of four closely spaced bands; mouse tau contains only three components with one of them being present in higher proportion than the others; adult guinea-pig tau is essentially present as a single band. 2. Microtubule-associated proteins were also prepared at different stages of brain development. In the three species only two bands were seen in the tau region at immature stages of development (fast tau and slow tau). However adult tau factors progressively replace the young entities. In contrast, only small changes were seen in the proportion of MAP2. 3. Peptide mapping analysis of the purified tau entities confirmed that the four adult rat proteins are very similar. In contrast, peptide mapping of the two young rat tau proteins were very different from each other and from those of the adult ones. Peptide mappings of young and adult MAP2 were only slightly different. 4. The activities of young tau proteins and young MAP2 in promoting pure tubulin assembly were much lower than those of the adult ones. Young fast tau and young slow tau were purified and both show to be active in promoting pure tubulin polymerization. 5. These data demonstrate the existence of two types of heterogeneity of microtubule-associated proteins: plurality of protein species at every stage of brain development and changes in composition and activity dependent on development.

Animals↗
PubMed1982

A biochemical model for neurite outgrowth during brain development.

Maximal amounts of tubulin in rat brain are observed during the 3 to 10-day postnatal period. The rates of in vitro tubulin polymerization are very low at these stages of development; they increase thereafter during the second postnatal week, reaching a maximum at adulthood. The increased rate of polymerization could depend either on modifications in the concentration and activity of microtubule-associated proteins (MAPs), which play a crucial role in microtubule assembly in vitro, or on changes in their composition. The results show that the composition and activity of TAU proteins (MW: 58-68000) change during development. Analysis of "young" and "adult" TAU protein peptide mapping suggests that their amino acid sequence is different. Our data indicate a good correlation between tubulin capacity to polymerize in vitro and changes in the composition and activity of TAU proteins which occur during the critical period when the neuronal network is constructed.

Aging↗
PubMed1981

Effects of early experience on the learning ability of yearling horses.

Twenty-four yearling Quarter Horse fillies were divided into three groups (I) very limited handling, (II) intermediate handling and (III) extensive handling. At about 14 months of age, each horse was preconditioned for 2 weeks and then run in a simple place-learning T-maze test in which it had to locate its feed. Thirty trials were run daily for 20 days, with the location of the feed changed each day. To retire from the maze, a horse had to meet the criterion: 11 correct responses in 12 tries, with the last eight being consecutive. Horses in Group II required the fewest trials to reach criterion. These horses also learned more and had the highest percentage of correct responses (P less than .05). Mean trainability tended to predict learning ability; however, trainability and trials to criterion were not significantly correlated. Mean emotionality scores indicated a tendency for horses in the intermediately handled group to be less emotional than those in Group I or III. Results indicated that horses with an intermediate amount of handling scored higher on an intermediate test of learning. All handled horses scored higher on learning tests than those not handled.

Animals↗
PubMed1980

Cytosolic thyroxine-binding protein and brain development.

The properties of cytosolic thyroxine binding protein were studied in the cortex and cerebellum of the rat at different stages of postnatal development: (1) Polyacrylamide-gel electrophoretic analysis showed that rat-brain cortex and cerebellum contain the same cytosolic thyroxine-binding protein which is very similar to the liver-corresponding entity. No changes in the electrophoretic mobility were seen during development in the 2 brain regions. In contrast, no defined triiodothyronine-binding component could be observed by the same technique. (2) Kinetic analysis studies revealed that the equilibrium of binding is reached in approximately 10 min whatever the brain region, the concentration of cytosolic protein and the stage of development. In all these cases saturation was obtained with the same thyroxine concentration (approximately 5 x 10(-7) M). Scatchard analysis also showed that whatever the experimental conditions, brain cytosolic protein contains a single class of thyroxine-binding sites with a K A of approximately 8 x 10(7) M-1. (3) Comparison of the K A during development showed that this constant remains unchanged from day 3 after birth until day 35 in both the cortex and the cerebellum. In contrast the number of binding sites significantly decreases in the cortex (approximately 2-fold; p less than 0.001) from day 3 to 35 with an already significant decline from day 3 to 6 (p less than 0.001). In the cerebellum this decline was even more marked since almost no binding activity was left at adulthood. Comparison of cortex and cerebellum binding activities also showed that this latter region contains approximately half the binding sites (p less than 0.001) at every stage of development studied.

Aging↗
PubMed1980

Rat, mouse, and guinea pig brain development and microtubule assembly.

The development of in vitro microtubule assembly and of tubulin concentration have been studied during brain maturation in the mouse and the rat, two species which have postnatal brain development, and in one species which is mature at birth, the guinea pig. (a) The rat of tubulin assembly is very slow soon after birth in both the mouse and rat; it increases progressively with age until adulthood. In contrast, in the guinea pig this rate is maximal at birth and slower rates are seen only at foetal stages. (b) Postnatal changes in the lag period of assembly and in the minimal concentration of tubulin (Cc) required to obtain in vitro assembly are seen in the mouse and the rat; in contrast these parameters are constant at all postnatal stages in the guinea pig with longer lag periods and lower Cc values being seen only at foetal stages. (c) Maximal rates of assembly, minimal lag periods, and minimal Cc values are restored after addition of microtubule-associated proteins to foetal guinea pig or young mouse and rat preparations, suggesting that the difference in the kinetic parameters of assembly between these species depends on differences in the concentration or activity of these proteins. (d) Maximal tubulin concentrations are observed before birth in the guinea pig and approximately at day 10 in the rat and mouse.

Aging↗
PubMed1979

Competitive inhibition of colchicine binding to tubulin by microtubule-associated proteins.

Microtubule-associated proteins (MAPs) promote tubulin polymerization, whereas colchicine inhibits this process. In this paper, MAPs have been shown to inhibit colchicine binding to tubulin in a competitive manner. Attempts were made to identify which of the MAPs fraction(s) was responsible; both tau protein (a thermostable molecule with a molecular weight of approximately 70,000) and a high molecular weight fraction (HMW) were able to compete with colchicine. In contrast, Mg2+, which also induces microtubule assembly in vitro, had no effect on colchicine binding to tubulin.

Animals↗
PubMed1979

Thyroid hormones and neurotubule assembly in vitro during brain development.

A new model has been used to evaluate the effects of thyroid hormones on brain development. This model is based on the assumption that the major effect of thyroid hormones is in regulating the rate of neurite growth of the rat brain at early stages of postnatal development. Microtubules were chosen as markers of neurite growth. We tested, therefore, whether the rate of microtubule assembly in vitro is under thyroid hormone control. The following results were obtained: The rate of tubulin assembly into microtubules in vitro seems to be thyroid hormone dependent: (a) in 15-day-old hypothyroid rats the rates of tubulin assembly in vitro are low, comparable to those levels found in normal rats on day 3; (b) normal rates of assembly in vitro are restored upon addition of very small amounts of microtubule fragments which act as nucleating centers in the process of microtubule formation; (c) addition of microtubule-associated proteins to a hypothyroid preparation restores maximal assembly rates; similar results were obtained on adding one of the microtubule-associated proteins (purified tau protein); (d) physiological amounts of thyroid hormones completely restore normal assembly rates provided that they are administered very early after birth; (e) the ability of tubulin to assemble maximally does not seem to be permanently impaired, since normal assembly rates are spontaneously restored when hypothyroidism is maintained until an adult stage; (f) normal microtubule assembly is observed when hypothyroidism is produced at an adult stage. The model which may be constructed from these results implies that thyroid hormones are required briefly after birth to accelerate the rate of microtubule assembly thus allowing intensive neurite growth during the critical period of brain development.

Aging↗
PubMed1977

Microtubule assembly in vitro. Purification of assembly-promoting factors.

The role of microtubule-associated proteins in the assembly of tubulin to microtubules in vitro has been studied. 1. It has been confirmed that pure tubulin obtained by phosphocellulose column chromatography does not significantly assemble in vitro in the absence of minor components which co-polymerize with tubulin. Although tubulin aggregates in a morpholino-ethanesulfonate buffer containing high Mg2+ concentrations, this process was neither inhibited by Ca2+ or colchicine, nor reversed by cold exposure. 2. Microtubule-associated proteins were prepared, either by phosphocellulose column chromatography or by a direct method based on boiling reassembled microtubules in the presence of 2 mM dithiothreitol and 0.75 M NaCl. From each of these preparations two protein fractions were purified, either by Ultrogel ACA34 chromatography or by sucrose gradient ultracentrifugation. The first one, with a high molecular weight, did not promote tubulin assembly; ageing of this material did not induce any activity. On the other hand, the second fraction, with an apparent molecular weight of 70 000 (tau protein), when almost completely purified, was active in promoting assembly. Thus a single specific protein is able to promote assembly of pure tubulin.

Animals↗