Biomedical subjects

L Denoroy

Publications and source records attributed to L Denoroy.

At least 73 records · Page 4Linked to original sources

PubMed1992

Elliptocytogenic alpha I/36 spectrin Sfax lacks nine amino acids in helix 3 of repeat 4. Evidence for the activation of a cryptic 5'-splice site in exon 8 of spectrin alpha-gene.

Elliptocytogenic alpha I/36 spectrin Sfax is a new variant found in a Tunisian family. The alpha I/36 allele yielded a clinically manifest picture only when occurring in trans to a recently identified, low expression level polymorphism referred to as the alpha V/41 allele. Spectrin dimers were slightly increased in 4 degrees C extracts. On peptide maps, the alpha I domain split into two abnormal fragments of 36 and 33 Kd. The mutated alpha-chain represented 20% and 44% of total alpha-chain in alpha/alpha I/36 and alpha V/41/alpha I/36 heterozygotes, respectively. Peptide sequencing showed that the 36-Kd fragment started at Ala 357 and displayed a deletion extending from amino acids 363 to 371. The corresponding 27-nucleotide deletion was found in alpha-spectrin mRNA. However, exon 8 of spectrin alpha-gene failed to disclose this deletion. Instead, an A to G substitution appeared in position 3 of codon 362, leading to the occurrence of the critical GU dinucleotide within a cryptic 5'-splice site surrounding codon 362. This event would account for the splicing out of codons 363 to 371. The reading frame was preserved and even amino acid 362 (AGG, Arg) remained unaltered. As in most spectrin alpha-chain elliptocytogenic variants, the change involved a helix 3. This is the first elliptocytogenic mutation recorded in repeat alpha 4.

Adult↗

PubMed1992

Effects of chronic atropine administration on regional vasoactive intestinal polypeptide concentrations in rat brain.

We studied the effects of 14 days' treatment with atropine sulfate (10 or 20 mg/kg per day) or atropine methyl bromide (20 mg/kg per day) on the concentration of vasoactive intestinal polypeptide like-immunoreactivity (VIP-LI) in the rat brain. VIP-LI in the anterior pituitary as well as brain areas dissected from treated and control rats was measured by radioimmunoassay. VIP-LI in the hypothalamus, and especially in the suprachiasmatic nucleus, was not affected by chronic atropine sulfate administration. Conversely, the same treatment induced a decrease in VIP-LI in the cerebral cortex, dorsal raphe, locus coeruleus, ventrolateral and dorsolateral medulla. In these structures, the decrease in VIP-LI was probably due to muscarinic receptor blockade in the central nervous system rather than in the peripheral nervous system since variations in VIP-LI were not observed after atropine methyl bromide treatment. These findings suggest the existence of a muscarinic control of VIP-LI in discrete brain areas of the rat and particularly in caudal brainstem structures.

Animals↗

PubMed1992

Quantitative autoradiographic study of somatostatin and neurotensin binding sites in medulla oblongata of SIDS.

Quantitative autoradiography analysis of neurotensin (NT) and somatostatin (SS) binding sites was performed on coronal sections of the medulla oblongata from 2 fetuses, 6 controls and 7 victims of Sudden Infant Death Syndrome (SIDS). Throughout the first postnatal year, mean SS binding site density was similar in controls and SIDS in all structures of the medulla oblongata. The density of neurotensin binding sites was significantly higher in the nucleus of tractus solitarius (NTS) of SIDS than in controls, but there was no significant differences in the other areas of the medulla oblongata. Our findings suggest an immature developmental pattern of increased NT binding sites the NTS of SIDS. This alteration may be related to an abnormal central cardiorespiratory and arousal control which is thought to be present in SIDS.

Autoradiography↗

PubMed1992

Effect of paradoxical sleep deprivation on vasoactive intestinal peptide-like immunoreactivity in discrete brain areas and anterior pituitary of the rat.

To determine whether vasoactive intestinal peptide (VIP) is involved in paradoxical sleep (PS) homeostasis, VIP-like immunoreactivity (VIP-LI) of discrete brain areas was determined by radioimmunoassay after 24 and 48 h of PS deprivation by the watertank technique followed or not by 5 h of sleep rebound. This study was carried out with an environmental control (placed in dry watertank: DWC) and a nonstressed control. Such PS deprivation induced a decrease of VIP content in PS-deprived rats restricted to cortex and anterior pituitary. In the cortex, the decrease in VIP-LI was of the same magnitude after 24 and 48 h of PS deprivation and VIP-LI was normal by 5 h of sleep rebound; as such a decrease was also observed after 48 h in DWC, it could be due to the stress related to the experiment rather than lack of sleep. In the anterior pituitary, the decrease was related to the duration of deprivation with a greater decrease in VIP-LI after 48 than after 24 h of PS deprivation and specifically related to PS deprivation since it was not observed in DWC rats. After 5 h of sleep rebound, recovery of VIP-LI was total in the 24-h experiment and partial in the 48-h one. In all eight other structures studied, VIP was unchanged after experimentation. These results strongly suggest that VIP is not involved in PS homeostasis but as indicated by other experiments more probably in circadian organization of sleep.

Animals↗

PubMed1992

[Abnormalities of the nervous system in sudden infant death syndrome].

Anomalies seem to be present in the brain of infants who died of sudden infant death syndrome. They imply dendritic synapses, myelinisation, some neurotransmitters, some peptides, their enzymes and receptors. More precisely, catecholaminergic systems seem to be abnormal. The changes were found mainly in brain stem, in vegetative central areas. These anomalies are probably maturational in nature. They could represent a fragility of vegetative control systems, especially cardiorespiratory and swallowing control systems. They probably do not explain, per se, the occurrence of death; they probably would need to be potentiated by additional etiological factors (environmental, viral, etc). These anomalies have to be considered as provisional and, in our opinion, need confirmation. Apart from the brain stem, other areas are under investigation, particularly cerebral structures implied in temperature control, sleep-wakefulness control and hemispheric structures.

Brain↗

PubMed1991

Identification of F0 subunits in the rat liver mitochondrial F0F1-ATP synthase.

In order to identify the subunits constituting the rat liver F0F1-ATP synthase, the complex prepared by selective extraction from the mitochondrial membranes with a detergent followed by purification on a sucrose gradient has been compared to that obtained by immunoprecipitation with an anti-F1 serum. The subunits present in both preparations that are assumed to be authentic components of the complex have been identified. The results show that the total rat liver F0F1-ATP synthase contains at least 13 different proteins, seven of which can be attributed to F0. The following F0 subunits have been identified: the subunit b (migrating as a 24 kDa band in SDS-PAGE), the oligomycin-sensitivity-conferring protein (20 kDa), and F6 (9 kDa) that have N-terminal sequences homologous to the beef-heart ones; the mtDNA encoded subunits 6 (20 kDa) and 8 (less than 7 kDa) that can be synthesized in isolated mitochondria; an additional 20 kDa protein that could be equivalent to the beef heart subunit d.

Adenosine Triphosphatases↗

PubMed1991

Descending adrenergic input to the primate spinal cord and its possible role in modulation of spinothalamic cells.

The present study focuses on 3 different aspects of the descending adrenergic system in the primate: (1) the distribution of adrenergic fibers and terminals in the spinal cord, (2) the source of this input and (3) the possible physiological effects of this system on spinal nociceptive processing. Antibodies to the enzyme phenylethanolamine-N-methyltransferase (PNMT) were employed to map the distribution of epinephrine-containing axonal profiles in the primate spinal cord. Smooth longitudinally oriented fibers were localized to the outer edge of the lateral funiculus. PNMT-containing axonal enlargements were distributed to the superficial dorsal horn, intermediate gray matter and the region surrounding the central canal at all spinal cord levels. PNMT-immunostained profiles were also observed in the intermediolateral cell column. A double labeling study employing retrograde transport of HRP from the spinal cord and PNMT immunohistochemistry identified a small population of HRP-PNMT-labeled neurons in the 'C1' region at the levels of the medulla and ponto-medullary junction. Thus, these cells are a probable source of adrenergic input to the spinal cord. Electrophysiological studies demonstrated that iontophoresis of epinephrine onto identified primate spinothalamic tract neurons in the lumbar dorsal horn resulted in inhibition of the glutamate-induced firing of these cells. The data from these studies support the hypothesis that adrenergic (PNMT-containing) cells in the caudal brainstem project to all levels of the cord and may contribute to descending modulation of nociceptive processing at these levels.

Animals↗

PubMed1991

Daily variations in concentration of vasoactive intestinal polypeptide immunoreactivity in discrete brain areas of the rat.

The concentration of VIP-like immunoreactivity (VIP-LI) was determined by radioimmunoassay at various times of the day in 11 discrete brain areas and in the antehypophysis of rats maintained on a regular 12 h/12 h light-dark cycle. In the suprachiasmatic and periventricular nuclei the VIP-LI increased during the dark period and decreased during the subsequent light period. In addition, the VIP-LI exhibited complex variations in the locus coeruleus, the periaqueductal gray matter and the paraventricular nucleus, but did not change in the other areas investigated.

Analysis of Variance↗

PubMed1991

Use of a fast protein electrophoretic purification procedure for N-terminal sequence analysis to identify S-locus related proteins in stigmas of Brassica oleracea.

In the cruciferous plant Brassica oleracea L. (cabbage), the S-locus specific glycoproteins (SLSGs) isolated only in stigmas are considered to play an important role in the normal prevention of self-fertilization. Recent molecular data have shown that the gene encoding these glycoproteins (the SLG gene) belonged to a multigenic family consisting of about 10 homologous copies among which another member is expressed, the S-locus related gene (SLR1gene). Our aim was to determine whether the SLR1-gene proteins were expressed in the stigmatic tissues. We first identified the putative SLSGs or SLR1-proteins by Con A-peroxidase detection of glycoproteins separated after isoelectric focusing in polyacrylamide gels. We describe a fast purification procedure for the glycoproteins of interest, based on analytical isoelectric focusing, electrophoresis, and electroblotting of proteins onto polyvinylidene difluoride membranes. Blotted proteins were sequenced for N-terminal amino acid determination. By comparison of the N-terminal sequences of the purified proteins with the peptide sequence predicted from the SLR1-cDNA, we demonstrate the expression of SLR1-like proteins in stigmas of B. oleracea.

Amino Acid Sequence↗

PubMed1991

[Capillary electrophoresis: principle and applications].

Capillary Zone Electrophoresis is a new separation technique which is performed in capillaries (ID smaller than 100 microns). The separations are carried out according to several modes which are mainly the Free Solution Capillary Electrophoresis, the Micellar Electrokinetic Capillary Chromatography and the Gel Capillary Electrophoresis. Many detection systems can be used. This technique has many advantages: high separative power, short time of analysis, low cost of operation and very low sample consumption. Capillary electrophoresis and HPLC appear to be complementary because they are based on different physical and chemical properties. Capillary electrophoresis has a great potential of applications for both chemical and biochemical analysis. This new and very efficient analytical technique is of growing interest for biochemical analysis and its use is likely to increase rapidly.

Biological Assay↗

PubMed1990

Application of preparative high-performance liquid chromatography to the purification of a fetal ovine insulin-like growth factor II: N-terminal sequence determinations using two different carriers.

A highly efficient procedure for the purification to homogeneity of an ovine fetal insulin-like growth factor II (IGF II) is described. Fetal sheep serum was used as the source material, and the bioactivity was followed throughout purification by an IGF II radioreceptor assay. Ovine IGF II was isolated by a combination of gel permeation, ion-exchange chromatography and reversed-phase high-performance liquid chromatography. The amino-terminal sequence of the first 36 amino acid residues was compared using two supports (polyethylenimine and polybrene) as carrier for protein sequencing. Ovine fetal IGF II was found to differ from human IGF II in three residues of the C-domain, with serine, isoleucine and asparagine substituted for alanine, valine and serine, respectively, at positions 32, 35 and 36. The final yield of highly purified ovine fetal IGF II was 134 micrograms, starting from 450 ml of serum.

Amino Acid Sequence↗

PubMed1990

Isolation and structure elucidation of a novel 5-kDa peptide from neurohaemal lobes of the corpora cardiaca of Locusta migratoria (Insecta, Orthoptera).

Two predominant peptides have been isolated from neurohaemal lobes of corpora cardiaca of 8000 adults of Locusta migratoria. Both peptides have been unambiguously characterized by automated peptide microsequencing and liquid secondary-ion mass spectrometry as a 50-residue peptide (5K peptide) and a 48-residue isologue (5K' peptide). Computer search of sequence data banks did not reveal any significant similarity with other identified proteins. The 5K peptides are remarkably rich in alanine residues (25%) and contain a stretch of five consecutive alanines. This structure suggests that these molecules could correspond to spacer peptides. This assumption is corroborated in the accompanying paper [Lagueux et al. (1990) Eur. J. Biochem. 187, 249-254] on the molecular cloning of the precursor protein which attributes to the 5K peptides a role analogous to that of the C peptides of insulins.

Amino Acid Sequence↗

PubMed1990

Two elliptocytogenic alpha I/74 variants of the spectrin alpha I domain. Spectrin Culoz (GGT----GTT; alpha I 40 Gly----Val) and spectrin Lyon (CTT----TTT; alpha I 43 Leu---Phe).

Spectrin alpha I/74 elliptocytosis results from abnormalities involving the "head" region of spectrin dimer. Increased susceptibility to trypsin enhances cleavage of the alpha spectrin chain, yielding an increased amount of the alpha I 74-kD fragment at the expense of the alpha I 80-kD parent fragment. Recently we showed that the mutations causing the Sp alpha I/74 abnormality may lie in the alpha- or the beta-chain, and that spectrin Culoz and spectrin Lyon were two (alpha I/74) alpha-variants, respectively. We now show that the spectrin Culoz alpha I domain undergoes prominent tryptic cleavage after Lys 42, whereas cleavage prevails after Arg 39 in spectrin Lyon. Applying the polymerase chain reaction (PCR) technique to exon 2 of the spectrin alpha I domain, we have established that the mutation responsible for spectrin Culoz is alpha I 40 Gly----Val; GGT----GTT. Applying the PCR technique to the cDNA derived from reticulocyte mRNA, we have shown that the mutation responsible for spectrin Lyon is alpha I 43 Leu----Phe; CTT----TTT. Studies of normal controls and of family members using dot blot hybridization with allele-specific oligonucleotide probes confirmed these results. Variants such as spectrin Culoz and spectrin Lyon should provide insight into a region that participates in spectrin dimer self-association and whose susceptibility to proteolysis must reflect subtle conformational changes.

Amino Acid Sequence↗

PubMed1989

Comparative immunocytochemical study of the catecholaminergic and peptidergic afferent innervation to the dorsal vagal complex in rat and guinea pig.

Light and electron microscopic immunocytochemistry was used to study the fine structural organization of the catecholaminergic and hypothalamic peptidergic innervation of the dorsal vagal complex of the medulla oblongata in the rat and guinea pig, the latter of which is known to lack central adrenergic neurons. In the rat, adrenergic fibers immunoreactive to phenylethanolamine-N-methyltransferase were concentrated in the dorsal motor nucleus of the vagus, where they established frequent symmetric synapses with dendrites and perikarya. On the other hand, the density of both oxytocin- and corticotropin-immunoreactive fibers appeared far lower in this nucleus than in the dorsal regions of the nucleus of the tractus solitarius, where they formed asymmetric synapses with small dendrites. In tissue treated for the dual labeling of two neuronal antigens, oxytocin- or corticotropin-reactive fibers were in close contact with adrenergic neurons in this dorsal medullary region. In the guinea pig, unlike the rat, the dorsal motor nucleus of the vagus contained large amounts of oxytocin- and corticotropin-reactive fibers, which formed many symmetric synapses with perikarya and dendrites. Taken together, these data suggest that the control of vagal preganglionic neurons by hypothalamic peptidergic neurons involves a bisynaptic neuronal pathway including adrenergic medullary neurons in the rat, whereas it is direct in the guinea pig, which lacks this adrenergic relay.

Adrenergic Fibers↗

PubMed1989

Adrenergic input from medullary ventrolateral C1 cells to the nucleus raphe pallidus of the cat, as demonstrated by a double immunostaining technique.

By means of a double immunostaining technique using unconjugated cholera-toxin B subunit (CTb) as a retrograde tracer combined with phenylethanolamine-N-methyltransferase (PNMT) immunohistochemistry, we demonstrated that the nucleus raphe pallidus of the cat receives a major projection from the ventrolateral part of the rostral medulla corresponding to the nucleus paragigantocellularis lateralis and the ventrolateral medullary reticular formation just caudal to it. We further showed that nearly 60% of the total CTb-labeled cells in this region are immunoreactive to PNMT. These double-labeled cells constitute one-third of the total PNMT-immunoreactive cells.

Adrenergic Fibers↗

PubMed1989

N-terminal sequence of the rat liver beta-subunit in the mitochondrial ATPase-ATPsynthase.

The N-terminal amino acid residues of the beta-subunit in the rat liver mitochondrial ATPase - ATPsynthase have been identified by direct microsequencing after electrophoresis of either purified F1 or F0F1. The mature rat liver beta-subunit begins by two alanine residues that precede the glutamine recently proposed as the first amino acid of the sequence (Boulet, D., Poirier, J. and Côté, C., 1989, Biochem. Biophys. Res. Commun. 159, 1184-1190). This result indicates that the proteolytic cleavage of the beta-subunit precursor may occur at the level of this first alanine. This may be important in the understanding of proteolytic processing events which lead to the assembly of the ATPase-ATPsynthase subunits during mitochondrial biogenesis.

Amino Acid Sequence↗

PubMed1989

Distribution of phenylethanolamine N-methyltransferase cell bodies, axons, and terminals in monkey brainstem: an immunohistochemical mapping study.

Adrenaline (epinephrine) is an important candidate transmitter in descending spinal control systems. To date intrinsic spinal adrenergic neurons have not been reported; thus adrenergic input is presumably derived from brainstem sites. In this regard, the localization of adrenergic neurons in the brainstem is an important consideration. Maps of adrenergic cell bodies and to a lesser extent axons and terminal fields have been made in various species, but not in monkeys. Thus, the present study concerns the organization of adrenergic systems in the brainstem of a monkey (Macaca fascicularis) immunohistochemically mapped by means of an antibody to the enzyme phenylethanolamine N-methyltransferase (PNMT). PNMT-immunostained cell bodies are distributed throughout the medulla in two principal locations. One concentration of labeled cells is in the dorsomedial medulla and includes the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (X), and an area ventral to X in a region of the reticular formation (RF) known as the central nucleus dorsalis (CnD) of the medulla. A few scattered cells are observed in the periventricular gray just ventral to the IVth ventricle and on midline in the raphe. The second major concentration of PNMT-immunostained cells is located in the ventrolateral RF, lateral and dorsolateral to the inferior olive (IO), including some cells in the rostral part of the lateral reticular nucleus (LRN). Terminal fields are located in the NTS, X, area postrema (AP), and the floor of the IVth ventricle in the medulla and pons. A light terminal field is also observed in the raphe, particularly raphe pallidus (RP). A heavy terminal field is present in locus coeruleus (LC). Fibers labeled for PNMT form two major fiber tracts. One is in the dorsomedial RF extending as a well-organized bundle through the medulla, pons, and midbrain. A second tract is located on the ventrolateral edge of the medulla and caudal pons. Fibers in this tract appear to descend to the spinal cord. A comparison with maps of other catecholamine neurons in primates is discussed, confirming that the distribution of the adrenergic system in monkeys is similar to that described in the human.

Adrenergic Fibers↗

PubMed1989

Spectrin Tunis (Sp alpha I/78), an elliptocytogenic variant, is due to the CGG----TGG codon change (Arg----Trp) at position 35 of the alpha I domain.

Spectrin Tunis (Sp alpha I/78) is an alpha l domain variant that causes asymptomatic elliptocytosis in the heterozygote state. It is manifested by a reduction of spectrin dimer self-association and by the development of a major 78-Kd fragment at the expense of the alpha l 80-Kd fragment upon spectrin-limited digestion. Amino acid sequence analysis, following peptide transfer onto Immobilon membranes, showed that the 78-Kd fragment results from a sensitized cleavage after lysyl residue 10. Using a 13.5-kb genomic alpha-spectrin probe and the Xbal, Pvull, and Mspl polymorphic sites detected with this probe, we concluded that spectrin Tunis is associated with the + - + haplotype (in the above order). Twenty mer oligonucleotides, complementary to genomic segments from introns 2 and 3, respectively, were synthesized. We then performed DNA amplification and sequencing. In the two investigated carriers of spectrin Tunis, we found the C----T base substitution of the codon corresponding to position 35 of the alpha l domain (CGG----TGG; Arg----Trp). The mutation lies in the last part of an alpha helix that extends from residues 9 to 44 of partial repeat alpha 1' and is comparable with helix 3 of full repeats 1 to 5. The modified proteolytic site, located 25 amino acid residues upstream, occurs at the beginning of the helix.

Amino Acid Sequence↗