Biomedical subjects

L Denoroy

Publications and source records attributed to L Denoroy.

At least 37 records · Page 2Linked to original sources

PubMed1996

In vivo monitoring of extracellular noradrenaline and glutamate from rat brain cortex with 2-min microdialysis sampling using capillary electrophoresis with laser-induced fluorescence detection.

The measurement of neurotransmitters by capillary electrophoresis (CE) has emerged as a reliable and sensitive method for microdialysis sample analysis. This paper describes a method which employs laser-induced fluorescence detection (LIFD) of catecholamines and excitatory amino acid derivatives formed after reaction with naphthalene-2,3-dicarboxaldehyde. On-line derivatization of very small volumes of microdialysis samples (500 nl) is developed before two off-line analyses (total run time of less than 10 min) are performed to detect derivatives of catecholamines and excitatory amino acids formed in each sample. High microdialysis temporal resolution is reached (2-min fractions) for the simultaneous monitoring of noradrenaline (NA) and glutamate concentrations from rat brain cortex microdialysates. The system performance is evaluated and pharmacological characterization of the determination of NA in cortical dialysates by CE-LIFD is reported.

Animals↗

PubMed1996

Enhanced temporal resolution for the microdialysis monitoring of catecholamines and excitatory amino acids using capillary electrophoresis with laser-induced fluorescence detection. Analytical developments and in vitro validations.

This paper reports the development of a method based on capillary electrophoresis with laser-induced fluorescence detection for the simultaneous determination of catecholamines and excitatory amino acids on submicroliter microdialysis samples, with short analysis times (3 min or less), high sensitivity (nanomolar range, i.e., attomoles detected) and high separation efficiency (up to 1.10(6) theoretical plates). A continuous flow derivatization of small volumes of microdialysate (500 nl) using naphthalene-2,3-dicarboxaldehyde as derivatizing reagent is described. Thereafter, two subsequent off-line analyses are performed on each of the 30-s dialysates to determine catecholamines and amino acids. The performances of the present method are demonstrated in vitro by monitoring rapid fluctuations in the concentration of catecholamines and amino acids in the external microdialysis medium.

Catecholamines↗

PubMed1996

Alteration in central and peripheral substance P- and neuropeptide Y-like immunoreactivity after chronic hypoxia in the rat.

The influence of long-term hypoxia on substance P (SP) and neuropeptide Y (NPY)-like immunoreactivity (LI) in discrete brain areas and peripheral structures was assessed by radioimmunoassay. Rats were exposed to normobaric hypoxia (10% O2 in nitrogen) for 14 days. In the carotid bodies of hypoxic animals, NPY-LI was significantly increased (56% vs. normoxic controls) while SP-LI was unchanged. In the brain, NPY-LI was increased in the ventrolateral medulla oblongata (23%) and in the striatum (53%); however, SP-LI was unaltered in these two regions. In the anterior pituitary, NPY-LI was increased (99%), while SP-LI was decreased (37%). No significant alteration in NPY-LI and SP-LI was observed in other discrete brain areas or peripheral structures studied. These results show that, in the rat, long-term hypoxia induces changes in NPY-LI or SP-LI in a few central and peripheral structures; these biochemical alterations may be linked to adaptative mechanisms involving morphological changes in carotid bodies or alterations in sympathetic control and neuroendocrine function.

Adrenal Glands↗

PubMed1996

The primary structure of rat ribosomal protein L10: relationship to a Jun-binding protein and to a putative Wilms' tumor suppressor.

The amino acid sequence of the rat 60S ribosomal subunit protein L10 was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed by determination of the NH2-terminal amino acid sequence in the protein. Ribosomal protein L10 has 213 amino acids (the NH2-terminal methionine is removed after translation of the mRNA); the molecular weight is 24,456. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 8 to 10 copies of the L10 gene. The mRNA for the protein is about 900 nucleotides in length. Rat L10 is related to ribosomal proteins from other eukaryotes. Ribosomal protein L10 is, in addition, the mammalian homolog of the chicken Jun-binding protein and is nearly identical to a putative Wilms' tumor suppressor. This is a presumptive example, of which there are many others, of an extraribosomal function of a ribosomal protein.

Amino Acid Sequence↗

PubMed1996

The cisternae decorating the red blood cell membrane in congenital dyserythropoietic anemia (type II) originate from the endoplasmic reticulum.

We studied 20 individuals from 17 unrelated families with congenital dyserythropoietic anemia (type II; CDAII). The clinical phenotype was mild to moderate. The inheritance pattern was invariably recessive. Coomassie blue stained gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) show that band 3 was thinner and migrated slightly faster than usual. In addition, staining showed two unknown minor bands (in the patients), but not in normal controls, the obligate carrier parents, or in patients with other anemic syndromes. These minor proteins were studied using partial digestion, amino acid sequencing, Western blotting, immunofluorescence, and immunogold electron microscopy. They were identified as the glucose-regulated protein GRP78 and calreticulin that are resident proteins of the endoplasmic reticulum (ER). Using specific antibody, we showed that protein disulfide isomerase (PDI), a third major protein of the ER, was also present on the SDS-PAGE of red blood cell (RBC) ghosts. Immunofluorescence colocalized PDI with the dense discontinuous ring decorating the RBC membrane. Immunogold electron microscopy showed that PDI was localized in the lumen of the cisternae, confirming that these originate from the smooth ER. From a practical point of view, screening the above minor proteins in RBC membranes appears to be a straightforward and reliable diagnostic test for CDAII.

Amino Acid Sequence↗

PubMed1996

High-speed separation of subnanomolar concentrations of noradrenaline and dopamine using capillary zone electrophoresis with laser-induced fluorescence detection.

Capillary zone electrophoresis with laser-induced fluorescence detection has been shown to give rapid separations with high resolution and sensitivity. These advantages are documented for catecholamines analysis in the present work, which shows that separation of subnanomolar concentrations of dopamine and noradrenaline (detection limits of 8.6 x 10(-11) M, corresponding to a detected amount of 143 zeptomoles of each catecholamine) can be performed in 82 s with an efficiency of several million theoretical plates.

Dopamine↗

PubMed1996

Endosulfine, endogenous ligand for the sulphonylurea receptor: isolation from porcine brain and partial structural determination of the alpha form.

Anti-diabetic sulphonylureas act via high affinity binding sites coupled to K-ATP channels. Endosulfine, an endogenous ligand for these binding sites, was shown to exist in two molecular forms, alpha and beta, in both the pancreas and the central nervous system. We describe here the isolation, and partial structural characterization of alpha endosulfine derived from porcine brains by means of a series of chromatography runs and gel electrophoresis. Porcine alpha endosulfine is a protein with a molecular mass of 13,196 daltons as determined by mass spectrometry and which is N-terminally blocked. Tryptic digestion followed by separation of the fragments by HPLC and automated Edman degradation yielded a total of 72 amino acids in four partial sequences. Comparison of these sequences with that present in the National Biomedical Research Foundation protein data bank indicated a 82% identity with a 112-amino acid protein with a molecular mass of 12,353 daltons called "cyclic AMP-regulated phosphoprotein-19', isolated from the bovine brain as a substrate for protein kinase A.

ATP-Binding Cassette Transporters↗

PubMed1996

Pharmacological modulations of adrenergic phenotype in medullary C2 and C3 cell groups of adult rat.

The adrenergic phenotype was analysed in the rat's rostral dorsomedial medulla under normal conditions and 3 days after a single intraperitoneal injection of an eburnamine derivative, RU 24722, which increases tyrosine hydroxylase protein expression in the rostral portion of the nucleus tractus solitarius. This approach was investigated by a double immunofluorescence labelling of tyrosine hydroxylase and phenylethanolamine N-methyltransferase proteins. Under normal conditions, most adrenergic cell bodies are anatomically distributed in the dorsal and rostral medulla oblongata between the rostral part of the dorsal motor nucleus of the vagus nerve and the medial longitudinal fasciculus. Adrenergic neurons detected in this medullar region were distributed between both cell groups. Three days after the pharmacological RU 24722 treatment, an upregulation in tyrosine hydroxylase and phenylethanolamine N-methyltransferase protein expression was detected in both cell groups characterized by a highly increased number of tyrosine hydroxylase- and phenylethanolamine N-methyltransferase-containing cell bodies. The number of TH-mRNA containing neurons was also increased, indicating the transcriptional level of this regulation. These results demonstrated a particular neuronal plasticity of adrenergic phenotype in the medullary cell groups of adult rat.

Analysis of Variance↗

PubMed1995

Sequences of V kappa L subgroup light chains in Fanconi's syndrome. Light chain V region gene usage restriction and peculiarities in myeloma-associated Fanconi's syndrome.

Certain monoclonal Ig light chains (LC) are responsible for marked disturbances of proximal tubule cell functions (Fanconi's syndrome, FS). In patients with FS, intracellular crystal-like inclusions containing LC determinants are commonly found in plasma cells, macrophages, and renal tubular cells. In an attempt at understanding the pathogenesis of myeloma-associated FS, we recently determined the first complete primary sequence of a kappa-LC (CHEB) responsible for the disease. We now report on the primary structure of three other kappa-LC of the V kappa l variability subgroup associated with FS (TRE, TRO, and DEL). After PCR amplification, cDNA encoding these LC were sequenced. CHEB, TRE, and TRO LC genes were found to be highly homologous to the same germline gene O2/O12. These patients had numerous intracellular crystals, whereas the fourth patient, DEL, had no detectable crystals. The LC from the latter patient was homologous to another germline gene, O8/O18. Comparison of these LC sequences to previously reported LC of the V kappa l subgroup allowed identification of a number of unusual amino acid substitutions in the V region that had rarely or never been previously described at the corresponding positions. Some of these unusual substitutions affect highly conserved amino acids located either in an external loop (residue 30) or in inner (residues 48 and 55) and outer (positions 63 and 72) beta-sheets that may be important for the structure and binding properties of the kappa-chains. These and several other substitutions, some of them shared with amyloidogenic kappa-LC, could induce conformational alterations and represent a determinant pathogenic factor.

Amino Acid Sequence↗

PubMed1995

Do transmembrane segments in proteolyzed sarcoplasmic reticulum Ca(2+)-ATPase retain their functional Ca2+ binding properties after removal of cytoplasmic fragments by proteinase K?

The present study was undertaken to investigate the Ca2+ binding properties of sarcoplasmic reticulum Ca(2+)-ATPase after removal of the cytoplasmic regions by treatment with proteinase K. One of the proteolysis cleavage sites (at the end of M6) was found unexpectedly close to the predicted membrane-water interphase, but otherwise the cleavage pattern was consistent with the presence of 10 transmembrane ATPase segments. C-terminal membranous peptides containing the putative transmembrane segments M7 to M10 accumulated after prolonged proteolysis, as well as large water-soluble fragments containing most of the phosphorylation and ATP-binding domain. Ca2+ binding was intact after cleavage of the polypeptide chain in the N-terminal region, but cuts at other locations disrupted the high affinity binding and sequential dissociation properties characteristic of native sarcoplasmic reticulum, leaving the translocation sites with only weak affinity for Ca2+. High affinity Ca2+ binding could only be maintained when proteolysis and subsequent manipulations took place in the presence of a Ca2+ concentration high enough to ensure permanent occupation of the binding sites with Ca2+. We conclude that in the absence of Ca2+, the complex of membrane-spanning segments in proteolyzed Ca(2+)-ATPase is labile, probably because of relatively free movement or rearrangement of individual segments. Our study, which is discussed in relation to results obtained on Na+,K(+)-ATPase and H+,K(+)-ATPase, emphasizes the importance of the cytosolic segments of the main polypeptide chain in exerting constraints on the intramembranous domain of a P-type ATPase.

Amino Acid Sequence↗

PubMed1995

Capillary zone electrophoresis with laser-induced fluorescence detection for the determination of nanomolar concentrations of noradrenaline and dopamine: application to brain microdialysate analysis.

Determination of catecholamines by capillary zone electrophoresis with laser-induced fluorescence detection was performed on low-concentration samples, which were derivatized with naphthalene-2,3-dicarboxaldehyde to give highly fluorescent compounds. When the borate concentration in the derivatization medium was decreased from 130 to 13 mM, sensitivity for noradrenaline (NA) and dopamine (DA) was greatly enhanced while resolution between these two compounds decreased. A 50 mM borate concentration in derivatization medium was chosen since it provided maximal resolution between NA and DA, together with a high separation efficiency (3.1 million theoretical plates per meter for DA). The injection of 2.4 nL of a NA and DA solution derivatized at 10(-9) M produced peaks with signal-to-noise ratio of 8:1 and 3:1, respectively, corresponding to 1.8 amol of each catecholamine. The calibration curves were linear when NA and DA solutions were derivatized at concentrations ranging from 10(-6) to 10(-9) M. This method was used to determine NA in brain extracellular fluid: a peak corresponding to a basal level of 5 x 10(-9) M endogeneous NA was observed in microdialysates from the medial frontal cortex of the rat, and its nature was confirmed by both electrophoretic and pharmacological validations.

Animals↗

PubMed1995

Phosphorylation of ribosomal protein L30 after herpes simplex virus type 1 infection.

In addition to an irreversible stimulation of S6 ribosomal protein phosphorylation, there is a modification of a subset of ribosomal proteins by phosphorylation after herpes simplex virus type 1 (HSV-1) infection. Moreover, in the course of this infection, three additional phosphorylated proteins can be extracted from ribosomes and separated by two-dimensional electrophoresis (2-DE) of total ribosomal proteins. One of them exhibits an identical molecular mass to L30, while being more acidic. This protein is phosphorylated on serine residues. The kinetics of appearance of this protein in the ribosomal fraction correlated with a decrease in L30 staining, as shown by 2-DE. Determination of the N-terminal amino acid sequence of this extra phosphoprotein and of L30-derived peptides demonstrated the identity of these two proteins.

Amino Acid Sequence↗

PubMed1995

Elicitin produced by an isolate of Phytophthora parasitica pathogenic to tobacco.

Elicitin was prepared from a Phytophthora parasitica culture isolated from tobacco in Australia. The protein with elicitor activity was purified and sequenced. This protein, which contains 98 amino acid residues, shows full homology with an elicitin produced by a completely unrelated isolate from carnation, indicating conservation of elicitin sequence within a single species.

Amino Acid Sequence↗

PubMed1994

Creatine kinase compactness and thiol accessibility during sodium dodecyl sulfate denaturation estimated by resonance energy transfer and 2-nitro-5-thiocyanobenzoic acid cleavage.

We have investigated the effect of increasing sodium dodecyl sulfate (SDS) concentrations on rabbit muscle cytosolic creatine kinase structure by two methods. We have first determined the variation of accessibility of the thiol groups of the enzyme during SDS denaturation by a technique which involves an irreversible chemical modification of CK accessible thiol groups, followed by NTCB cleavage before the unmodified cysteines in 8 M urea (pH 9) and analysis of the peptides obtained by resolutive gel electrophoresis, without sequencing. We have determined that the order of accessibility of CK MM cysteine residues during SDS denaturation is Cys-282, Cys-145 and then Cys-253. The fourth cysteine residue, Cys-73, is never titrated even at high SDS/CK molar ratio. In contrast, the three last residues are simultaneously titrated when CK is denatured in guanidinium chloride. Thus, SDS-denatured CK seems to retain some residual organized structure. In order to confirm this hypothesis, compactness of the molecule was estimated by fluorescence energy transfer between CK tryptophans and AEDANS, an extrinsic fluorophore. The location of this fluorophore on the accessible thiol of Cys-282 was verified by the previous technique. The results of these experiments do indicate that SDS-denatured CK is more compact than CK completely unfolded in guanidinium chloride.

Animals↗

PubMed1994

Chronic hypoxia affects peripheral and central vasoactive intestinal peptide-like immunoreactivity in the rat.

The influence of long-term hypoxia on vasoactive intestinal peptide-like immunoreactivity (VIP-LI) in discrete brain areas and peripheral structures was assessed by RIA. Rats were exposed to normobaric hypoxia (10% O2-90% N2) for 14 days. VIP-LI was significantly increased in carotid bodies of hypoxic animals (204% vs. normoxic animals). On the other hand, VIP-LI was decreased in the anterior pituitary (-68%), suprachiasmatic nuclei (-29%) and periventricular nuclei (-26%). No significant variation in VIP-LI was observed in other peripheral structures and discrete brain area studied. These results suggest that long-term hypoxia induces alterations in VIP systems implicated in chemoreception, biological rhythms and neuroendocrine functions.

Animals↗

PubMed1994

Detection of neurotensin in tissues by capillary zone electrophoresis.

An electrophoretic method for detecting neurotensin in tissues was developed. Homogenates of rat duodenum and adrenal glands were first extracted by solid-phase extraction and purified by reversed-phase high-performance liquid chromatography. The neurotensin-rich fraction was further analyzed by capillary zone electrophoresis (CZE) using a commercial instrument with UV detection. A minor peak was detected as neurotensin and its identity was further confirmed by performing several CZE analyses at different pH values. Such an approach could be used to analyze with good molecular specificity the neuropeptides in some human tissues.

Adrenal Glands↗

PubMed1994

Overrepresentation of the V kappa IV subgroup in light chain deposition disease.

The variability subgroup of human monoclonal kappa chains purified from urine in 3 consecutive patients with myeloma associated light chain deposition disease was determined from amino acid sequences of their first framework regions (FR1). N-glycosylation was searched for by N-glycosidase F treatment. These data together with our previously published results, indicate the pathogenic potential of the rare V kappa IV subgroup and confirm the absence of detectable serum and urine free monoclonal light chains when they are N-glycosylated.

Amino Acid Sequence↗

PubMed1994

VIP binding sites in adult rat hypothalamus: nuclear distribution and daily variations.

Vasoactive intestinal peptide (VIP) neurons are present in the suprachiasmatic nucleus (SCN) and project to various hypothalamic nuclei. Several results suggest that hypothalamic VIP is involved in the mediation of synchronizing information. [125I]Vasoactive intestinal peptide binding has been reported to be especially high in the hypothalamus. We show here by radioautography by dipping that [125I]VIP binding is high and homogeneously distributed in the SCN. The correspondence between VIP binding and terminals densities supports the hypothesis that VIP acts through synapses in the SCN. Moderate and fairly homogeneous VIP binding is also found in the paraventricular nucleus (PaVN), whereas VIP terminals are reported to be restricted to the sub-paraventricular area. This suggests that VIP could diffuse and thus function as a paracrine neuromodulator in the PaVN. [125I]Vasoactive intestinal peptide binding is stable along the nycthemer in the suprachiasmatic, peri-, and paraventricular nuclei as well as in all other areas investigated.

Animals↗