Monique Rogard

Lhx9: A Novel LIM-Homeodomain Gene Expressed in the Developing Forebrain

A novel LIM-homeodomain gene, Lhx9, was isolated by degenerate RT-PCR followed by mouse embryonic library screening.Lhx9 cDNA encodes a protein that is most closely related to Drosophila apterous and rodent Lhx2 proteins. TheLhx9 spatiotemporal pattern of expression during embryogenesis was similar but distinct from Lhx2. Highest expression levels were found in the diencephalon, telencephalic vesicles, and dorsal mesencephalon. Domains of expression respected the proposed neuromeric boundaries (Puelles and Rubenstein, 1993).Lhx9 was also expressed in the spinal cord, forelimb and hindlimb mesenchyme, and urogenital system. AlthoughLhx9 expression was sustained in diencephalon and mesencephalon from embryonic day 10.5 (E10.5) to postnatal stages, it was transient in the future cerebral cortex, where it was turned off between E14.5 and E16.5. Lhx9 expression was highest if not exclusively located (depending on the region of interest) in the intermediate and mantle zones, as opposed to the mitotic ventricular zone. Lhx9 protein was tested for interaction with the recently discovered cofactors of LIM-homeodomain proteins and was found to interact strongly both with CLIM1 and CLIM2. The expression pattern and structural characteristics of Lhx9 suggest that it encodes a transcription factor that might be involved in the control of cell differentiation of several neural cell types. Furthermore, Lhx9 protein could act in a combinatorial manner with other LIM-homeodomain factors expressed in overlapping pattern.

Parallel Decrease of Glutamic Acid Decarboxylase and Preproenkephalin mRNA in the Rat Striatum Following Chronic Treatment with a Dopaminergic D1 Antagonist and D2 Agonist

Abstract: The levels of mRNA encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot analysis, in the dorsal and the ventral part of the striatum, following long‐term treatments with drugs acting selectively on D1 or D2 dopaminergic receptors. Chronic injection of the selective D1 antagonist SCH 23390 elicited a significant decrease in level of both GAD and PPE mRNA (−30%) in the dorsal striatum, whereas no significant change was observed in the ventral striatum. Chronic administration of both SCH 23390 and RU 24926, a D2 agonist, decreased the GAD and PPE mRNA levels in the dorsal (−38 and −57%, respectively) as well as in the ventral (−70 and −60%, respectively) striatum. In the ventral striatum the marked reduction of GAD mRNA levels was paralleled by a significant decrease of Vmax values of GAD enzymatic activity (−41%). These results suggest that the decrease in content of both GAD and PPE mRNA, promoted by the chronic blockade of D1 receptors, is mainly due to the action of dopamine acting on unaffected D2 receptors. Indeed, this decrease is further amplified when the D2 agonist and the D1 antagonist are administered together. Our results substantiate further the molecular mechanisms by which dopamine acts on different populations of GABAergic and enkephalinergic neurons in the two striatal regions examined.

Membrane lipid dynamics and enzymic activity in bovine adrenal cortex microsomes.

The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C16 as compared to the C2 region, while the rotational motion appeared to be the most hindered at the C7–C9 level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C16 region. The extent to which the acyl chain reacted to this perturbation at the C12 level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the 3-oxosteroid Δ5−Δ4-isomerase, the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the 3-oxo-Δ4-steroids in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant (2–5 kcal · mol−1) in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy (20 kcal · mol−1) was observed at temperature below 16°C at pH 7.5. A correlated change of the pKESapp as function of temperature was detected; at 36°C pKESapp = 8.3 while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the 3β-hydroxy-Δ5-steroid dehydrogenase, the 3-oxosteroid Δ5−Δ4-isomerase was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids.

Haloperidol increases Ppe mrna levels in the caudal part of the nucleus accumbens in the rat

Dopamine exerts a tonic inhibitory control in the regulation of mRNA encoding preproenkephalin (PPE), the precursor of enkephalin, in the dorsal part of the rat striatum. Less is known about the role of this amine in the regulation of PPE mRNA in the ventral part of the striatum which corresponds to the nucleus accumbens. In this study, the effects of an interruption of dopamine transmission by haloperidol (a dopaminergic receptor antagonist) were examined in the rat nucleus accumbens. The levels of PPE mRNA were analysed by in situ hybridization histochemistry by taking into account the complexity of the nucleus accumbens, i.e. by differentiating the rostro-caudal extension of the structure. It was found that PPE mRNA content was increased significantly only in the caudal part of the nucleus, indicating rostro-caudal differences in the haloperidol effects. The differential reactivity of PPE mRNA to dopaminergic receptor blockade and to a lesion of dopaminergic neurones along the rostrocaudal axis of the ventral striatum is discussed.

Effects of temperature and benzyl alcohol on the structure and adenylate cyclase activity of plasma membranes from bovine adrenal cortex.

Adenylate cyclase activation by corticotropin (ACTH), fluoride and forskolin was studied as a function of membrane structure in plasma membranes from bovine adrenal cortex. The composition of these membranes was characterized by a very low cholesterol and sphingomyelin content and a high protein content. The fluorescent probes 1,6-diphenylhexa-1,3,5-triene (DPH) and a cationic analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) were, respectively, used to probe the hydrophobic and polar head regions of the bilayer. When both probes were embedded either in the plasma membranes or in liposomes obtained from their lipid extracts, they exhibited lifetime heterogeneity, and in terms of the order parameter S, hindered motion. Under all the experimental conditions tested, S was higher for TMA-DPH than for DPH but both S values decreased linearly with temperature within the range of 10 to 40 degrees C, in the plasma membranes and the liposomes. This indicated the absence of lipid phase transition and phase separation. Addition to the membranes of up to 100 mM benzyl alcohol at 20 degrees C also resulted in a linear decrease in S values. Membrane perturbations by temperature changes or benzyl alcohol treatment made it possible to distinguish between the characteristics of adenylate cyclase activation with each of the three effectors used. Linear Arrhenius plots showed that when adenylate cyclase activity was stimulated by forskolin or NaF, the activation energy was similar (70 kJ.mol-1). Fluidification of the membrane with benzyl alcohol concentrations of up to 100 mM at 12 or 24 degrees C produced a linear decrease in the forskolin-stimulated activity, that led to its inhibition by 50%. By contrast, NaF stabilized adenylate cyclase activity against the perturbations induced by benzyl alcohol at both temperatures. In the presence of ACTH, biphasic Arrhenius plots were characterized by a well-defined break at 18 degrees C, which shifted at 12.5 degrees C in the presence of 40 mM benzyl alcohol. These plots suggested that ACTH-sensitive adenylate cyclase exists in two different states. This hypothesis was supported by the striking difference in the effects of benzyl alcohol perturbation when experiments were performed below and above the break temperature. The present results are consistent with the possibility that clusters of ACTH receptors form in the membrane as a function of temperature and/or lipid phase fluidity.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of haptoglobin in chicken serum and specificity of the chicken haptoglobin-hemoglobin complex formation

1. The presence of haptoglobin in chicken serum has been demonstrated by three different techniques: gel filtration, cellulose acetate electrophoresis and fluorescence quenching. 2. Chicken haptoglobin shows a narrow species specificity; it binds only avian and reptilian but not mammalian hemoglobins. 3. Haptoglobin seems to have been subjected to profound changes during the course of evolution.

The association equilibrium between haptoglobin and apohemoglobin

Abstract The binding of haptoglobin to apohemoglobin (hemoglobin devoid of heme) has been investigated. Haptoglobin of genetic type 1-1 labelled by 1-dimethylamino-naphthalene 5-sulfonyl chloride or 2-dimethylaminonaphthalene 5-sulfonyl chloride has been titrated with apohemoglobin in 20mM phosphate pH 6.8 or 5.7, at 5°C. The formation of the complex has been followed by the increase of the polarization fluorescence. The titration curves show that dansylated haptoglobin and apohemoglobin are in association equilibrium and that one haptoglobin molecule binds two molecules of apohemoglobin. The calculated association constant is K = 4.6 × 106.

Adenylate cyclase of bovine adrenal cortex plasma membranes: divergence between corticotropin and fluoride combined effects with forskolin

The diterpene forskolin maximally stimulated bovine adrenal cortex adenylate cyclase activity 9‐fold with a concentration producing half‐maximum effect (ED50) of about 4 μM. The effects of forskolin and the fully active corticotropin fragment ACTH (1–24) were additive over nearly the whole range of concentration of both effectors, indicating separate and independent mechanisms of action. By contrast, 10 mM NaF blocked forskolin action in the nanomolar range of the diterpene concentration, while it allowed a partial stimulation by forskolin in the micromolar range. NaF thus reveals a heterogeneity of forskolin action in the adrenal cortex plasma membranes. Moreover, our data suggest that ACTH and NaF activation effects, both mediated by the stimulatory regulatory protein Gs, proceed through different mechanisms.

Comparative Distribution of D1 Receptors and μ Opiate Receptors in the Neonatal Rat Striatum: Effects of an Early Lesion of Dopaminergic Fibers

The compartmental organization of the striatum in striosomes and matrix (Graybiel and Ragsdale, 1978) is followed by a large variety of neuronal markers. Peptide-containing fibers and peptide-containing perikarya (Graybiel and Ragsdale, 1983; Graybiel, 1984a), afferent fibers (Gerfen et al., 1987a; Gerfen, 1989; Jimenez-Castellanos and Graybiel, 1987) efferent neurons (Jimenez-Castellanos and Graybiel, 1989), are mostly distributed according to this two compartments. Various specific receptor ligands have also been shown to label binding sites organized according to a striosomal pattern, as for instance μ opiate receptors (Herkenham and Pert, 1981), muscarinic M1 receptors (Nastuk and Graybiel, 1985), benzodiazepine type II sites (Faull and Villiger, 1988). With regard to dopamine (DA) receptors, a compartmental distribution has been observed in the striatum of cat and monkey, with heightened density of Dl receptors (Besson et al., 1988) in striosomes and heightened density of D2 receptors in the matrix (Beckstead et al., 1988). No such heterogeneity has been described for Dl or D2 receptors in the striatum of the adult rat (Boyson et al., 1986; Savasta et al., 1986). However, it has been shown in feline (Richfield at al., 1987) and rodents (Murrin and Zeng, 1989; Ohta and al., 1989) that both striatal DA fibers and striatal D1 receptors are clustered during the neonatal period. The purpose of our study was to reexamine, in the rat striatum, the postnatal evolution of the distribution of Dl receptors, and to analyze the possible role of the incoming DA fibers in the disappearance of their patchy organization. The distribution of D1 receptors was compared to that of μ opiate receptors during the first three postnatal weeks in intact rats and in rats treated at postnatal day 2 (P2) by an intrastriatal 6-OHDA injection.

Inhibitory Role of Dopaminergic D2 Receptors in the Expression of Glutamic Acid Decarboxylase and Preproenkephalin mRNA in the Rat Striatum

Dopamine (DA) contained in the dense network of DA fibers innervating the striatum has been shown to play a major role in the regulation of striatal neuron activity. This has been approached by analyzing either changes in electrophysiological properties of neurons (Bernardi et al., 1978; Calabresi et al., 1988) or changes in biochemical parameters such as the levels and/or the synthesis (Hong et al., 1978a and b; Hanson et al., 1988; Li et al., 1987) or the release (Lehman and Langer, 1983; Girault et al., 1986a) of specific neurotransmitters localized in striatal neurons. A useful tool in understanding the regulation of neuronal activity is now offered by molecular biology which allows the identification of specific mRNAs, reflecting earlier events than neurotransmitter contents. In this study, we tried to elucidate the DA receptor subtype which mediates dopaminergic modulation of the expression of messengers encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE), two major markers contained in projection neurons of the striatum (Fonnum et al., 1974; Cuello et al. 1978). This analysis has been initiated by previous findings showing that a 6-hydroxydopamine (6-OHDA) lesion of nigrostriatal DA neurons produced increased levels of mRNAs encoding GAD (Vernier et al., 1988) and PPE (Angulo et al. 1986; Young et al., 1986; Normand et al. 1988), and that these effects could be reproduced by chronic treatments with haloperidol (Sabol et al., 1983; Tang et al., 1983; Sivam et al., 1986; Normand et al., 1987; Morris et al., 1988; Vernier unpublished observations). These previous studies suggested an inhibitory control of DA on the expression of both GAD- and PPE-mRNA. The action of DA being mediated by an interaction with at least two receptor subtypes, coupled to different second messenger systems (Stoof and Kebabian, 1981), it was of interest to elucidate the receptor subtype involved in these effects.