Biomedical subjects

S Raimbault

Publications and source records attributed to S Raimbault.

26 records · Page 2Linked to original sources

PubMed1992

Antibodies selected from whole antiserum by fusion proteins as tools for the study of the topology of mitochondrial membrane proteins. Evidence that the N-terminal extremity of the sixth alpha-helix of the uncoupling protein is facing the matrix.

The reactivity to freeze-thawed mitochondria or submitochondrial particles of a whole antiserum raised against the uncoupling protein has been investigated. Incubation with freeze-thawed brown adipose tissue mitochondria trapped antibodies reactive toward accessible parts of the uncoupling protein. One-third to one-half of antibodies against uncoupling protein which were present in the serum remained free. These antibodies were highly reactive with the vesicles obtained by sonication of mitochondria, in which the matricial side of the inner membrane was made accessible. To define epitopes recognized by the antiserum, different fusion proteins made up of MalE protein and uncoupling protein fragments were used. Immunoaffinity chromatography, using an immobilized purified fusion protein containing amino acids 253 to 290 of uncoupling protein, selected antibodies specifically directed against this part of the protein. A more precise localization of the main epitope recognized by these antibodies is proposed. These purified antibodies reacted with the protein only in submitochondrial particles, indicating a matricial orientation of this epitope. This result, associated with other data concerning uncoupling protein or related mitochondrial carriers such as the ADP/ATP translocator and the phosphate carrier, allowed us to determine the orientation of the sixth alpha-helix of the uncoupling protein.

Amino Acid Sequence↗

PubMed1992

Comment to Shinohara et al. (1991) FEBS Letters 293, 173-174. The uncoupling protein is not expressed in rat liver.

Using Northern blot analysis, immunoblotting with purified antibodies and Polymerase Chain Reaction analysis, we were unable to detect the Uncoupling Protein-UCP or its mRNA in liver of control, cold-exposed or newborn rats. The unique expression of this protein in brown adipocytes was confirmed. These data refute the surprising recent report on UCP expression in rat liver (Shinohara (1991) FEBS Lett. 293, 173-174). Moreover we report that the hybridization signal obtained by these authors is probably non-specific and due to the 3' non-coding domain of the UCP cDNA probe.

Animals↗

PubMed1992

Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological characterization.

Brown adipocytes are thermogenic cells which play an important role in energy balance. Their thermogenic activity is due to the presence of a mitochondrial uncoupling protein (UCP). Until recently, it was admitted that in rodents brown adipocytes were mainly located in classical brown adipose tissue (BAT). In the present study, we have investigated the presence of UCP protein or mRNA in white adipose tissue (WAT) of rats. Using polymerase chain reaction or Northern blot hybridization, UCP mRNA was detected in mesenteric, epidydimal, retroperitoneal, inguinal and particularly in periovarian adipose depots. The uncoupling protein was detected by Western blotting in mitochondria from periovarian adipose tissue. When rats were submitted to cold or to treatment with a beta-adrenoceptor agonist, UCP expression was increased in this tissue as in typical brown fat. Moreover, the expression was decreased in obese fa/fa rats compared to lean controls. Morphological studies showed that periovarian adipose tissue of rats kept at 24 degrees C contained cells with numerous typical BAT mitochondria with or without multilocular lipid droplets. Immunocytochemistry confirmed that multilocular cells expressed mitochondrial UCP. Furthermore, the number of brown adipocytes and the density of mitochondrial cristae increased in parallel with exposure to cold. These results demonstrate that adipocytes expressing UCP are present in adipose deposits considered as white fat. They suggest the existence of a continuum in rodents between BAT and WAT, and a great plasticity between adipose tissue phenotypes. The physiological importance of brown adipocytes in WAT and the regulation of UCP expression remain open questions.

Adipose Tissue↗

PubMed1990

Expression of the brown fat mitochondria uncoupling protein in Xenopus oocytes and important into mitochondrial membrane.

Non shivering thermogenesis of brown adipose tissue is due to the uncoupling protein (UCP), located in the inner mitochondrial membrane, which functions as a proton translocator and can thus uncouple mitochondrial respiration. We describe here the expression of UCP in Xenopus laevis oocytes after injection of UCP mRNA, which was transcribed in vitro. UCP seems to be correctly transported into mitochondria and integrated into the membrane, but we were not able to establish definitely the functionality of this UCP. We conclude that this expression system could be suitable for the study of the mitochondrial import mechanism but not for the examination of physiological properties of UCP.

Adipose Tissue, Brown↗

PubMed1990

Human uncoupling protein gene: structure, comparison with rat gene, and assignment to the long arm of chromosome 4.

The uncoupling protein (UCP) gene encodes a unique mammalian mitochondrial proton carrier that induces heat production in brown adipocytes. Human UCP gene was isolated and its organization analyzed. A comparison was made with rat UCP gene. Human UCP gene spans 13 Kb and contains a transcribed region that covers 9 Kb of the human genome. All of the exons were also sequenced except the extreme end of the 3' untranslated region. Two Kb DNA upstream the TATA box were also sequenced. This region contains several fragments that are highly homologous to the gene of rat UCP. Neither CCAAT sequence nor Sp 1 binding motif were detected. Human UCP gene is split into six exons. The complete amino acid sequence of the protein was determined. Human UCP has 305 amino acids and a molecular weight of 32,786. It has no N-terminal targeting sequence. It is 79% homologous to rat UCP both at nucleotidic and amino acid levels. The primary structure of UCP is significantly homologous to the primary structure of the human T1 ADP/ATP carrier, particularly in the C-terminal extremity, which is supposed to contain a nucleotide-binding site in both proteins. Human UCP gene is single type, as it is in rodents. Two genomic fragments were used to detect a 1.9 Kb mRNA in human perirenal brown adipose tissue. Using in situ hybridization, UCP gene was assigned in humans to chromosome 4 in q31. Interestingly, the T1 gene encoding the heart-skeletal muscle ADP/ATP carrier has recently been shown to be on the same chromosome (Li et al. Biol Chem 264:13998, 1989).

Amino Acid Sequence↗

PubMed1990

Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells.

The mitochondrial uncoupling protein (UCP) is a membranous proton carrier exclusively synthesized in brown adipocytes. The cDNA for the rat UCP was placed in an expression vector and transfected into mammalian cells. Its expression was tested in transiently transfected CHO cells. In these cells the UCP was detected in mitochondria by using antibodies. Permanent expression of the UCP was achieved in stable transformed CHO cell lines. In these cells the UCP was characterized in mitochondrial membranes, by using antibodies and hydroxyapatite purification. The protein expressed in CHO cells displayed the functional characteristics of brown adipocyte UCP. It induced the uncoupling of respiration in isolated CHO mitochondria. The membrane potential of transformed mitochondria was also significantly lowered, as a result of the proton translocating activity of the UCP. GDP is known to inhibit the proton pathway in brown fat mitochondria. Addition of GDP to CHO mitochondria containing UCP resulted in a recoupling of respiration and an increase in membrane potential. Thus we conclude that functional UCP is expressed in CHO cells and that the insertion of the UCP alone in any mitochondria is sufficient to induce the uncoupling of respiration. This approach should allow studies on the structure-function relationship of the UCP and of several other related mitochondrial carriers.

Adipose Tissue, Brown↗

PubMed1988

The gene for rat uncoupling protein: complete sequence, structure of primary transcript and evolutionary relationship between exons.

The complete nucleotide sequence of rat Uncoupling Protein gene has been determined. 4.5 Kb of the 5'-flanking region have also been sequenced. The site of transcription start as well as 3'-end extremities were identified. Transcription unit spans 8.4 Kb and contains 6 exons and 5 introns. Uncoupling protein as well as related mitochondrial carriers such as ADP/ATP carrier and phosphate carrier has a triplicated structure and each repeat of Uncoupling Protein corresponds to 2 exons. Two gene duplications could explain the triplicated structure of UCP and the more recent event of duplication concerned exons III and V. Evidence for homology between Uncoupling Protein and Neurospora crassa ADP/ATP carrier at the gene level was also noticed.

Animals↗

PubMed1988

Detection of brown adipose tissue uncoupling protein mRNA in adult patients by a human genomic probe.

1. Studies on human brown adipose tissue require specific molecular probes. A human genomic library has been screened with a complementary DNA corresponding to the uncoupling protein (UCP) of rat brown adipose tissue mitochondria. 2. Two recombinant phages were isolated, carrying genomic sequences of human UCP. From them we have subcloned a 0.5 kilobase fragment. This fragment, H-Ucp-0.5, contained two intronic regions and two exonic regions. Exonic regions encoded a sequence of 84 amino acids which exhibited a strong homology with central domain at rat UCP. The organization of H-Ucp-0.5 was confirmed by SI mapping analysis. 3. A Southern analysis suggested that the gene is single type in the human, as it is in rodents. 4. In Northern analysis experiments, H-Ucp-0.5 detected a specific 1.8 kb mRNA in human brown adipose tissue obtained from six patients with phaeochromocytoma and from one patient with a hibernoma. This molecular probe is a new, sensitive and reliable tool with which to study human brown adipocytes.

Adipose Tissue, Brown↗