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Biomedical subjects

M Raymond

Publications and source records attributed to M Raymond.

At least 91 records · Page 5Linked to original sources
PubMed1991

Simultaneous identification of eight leucocyte subsets of human peripheral blood using three-colour immunofluorescence flow cytometric analysis.

The full analytical potential of flow cytometry has been exploited in order to identify a maximum of human peripheral blood leucocyte subsets in a single tube. For this purpose a mixture composed of six different monoclonal antibodies directly coupled to FITC or phycoerythrin or indirectly linked to the DuoChrome reagent via the biotin/streptavidin system was used. This combination of monoclonal antibodies and fluorochromes offered the possibility of simultaneously determining eight leucocyte subsets of human peripheral blood using a single laser beam. These subsets were T4 cells, bright and dim T8 cells, T null (CD3+ CD4- CD8-) cells, NK cells and B cells, polymorphonuclear cells and monocytes. This method is very easy to perform, can be applied to samples from other lymphoid tissues such as tonsils and is particularly useful when starting with a low number of cells.

Antigens, CD↗
PubMed1991

Worldwide migration of amplified insecticide resistance genes in mosquitoes.

In Culex pipiens, overproduction of nonspecific esterases is a common mechanism of resistance to organophosphate insecticides. The esterases are attributed to closely linked loci named A and B according to substrate preference, and overproduction of all esterases B is due to gene amplification. Distribution of electrophoretically distinct variants of overproduced esterases A and B is geographically restricted, with the exception of esterases A2 and B2, always found together throughout at least three continents. To determine whether this situation is due to migration or to a high mutation rate, esterase B structural genes and their flanking regions were compared by sequence and/or restriction fragment length polymorphism analysis. Whereas structural genes were similar, flanking regions of electrophoretically dissimilar esterases B varied considerably. In contrast, flanking sequences of esterases B2 from different geographical locations (Africa, Asia, North America) were identical. These results suggest that amplified esterase B2 genes originated from an initial event that has subsequently spread organophosphate insecticide resistance by migration.

Amino Acid Sequence↗
PubMed1990

Cell-specific activity of cis-acting regulatory elements in the promoter of the mouse multidrug resistance gene mdr1.

To define cis-acting elements implicated in transcriptional regulation of the mouse multidrug resistance gene mdr1, we have cloned and characterized the 5' end of the gene. Nucleotide sequence analysis identified TATA, GGGCGG, and CCAAT consensus sequence elements at positions -27, -47, and -83, respectively. The transcriptional activities of 5' deletion fragments from the promoter linked to a reporter gene were tested in mouse cell lines of different tissue origins shown to express different levels of endogenous mdr1 RNA. Sequences located between nucleotides -93 and +84 were able to confer basal promoter activity and cell specificity to the reporter gene. The addition to the basal promoter of sequences upstream of position -141 was found to up or down regulate the basal level of expression of the reporter gene in a cell-specific manner.

Animals↗
PubMed1990

Physical mapping, amplification, and overexpression of the mouse mdr gene family in multidrug-resistant cells.

The mouse mdr gene family consists of three distinct genes (mdr1, mdr2, and mdr3), for which we have isolated full-length cDNA clones. cDNA subfragments corresponding to discrete regions showing little sequence conservation among the three mdr genes were used as gene-specific DNA probes in hybridization experiments. Long-range mapping by pulse-field gel electrophoresis indicated that the three mdr genes are closely linked on a genomic DNA segment of approximately 625 kilobases. The gene order and direction of transcription of the three genes were determined and indicate the arrangement (5') mdr3 (3')-(5') mdr1 (3')-(3') mdr2 (5'). Southern blotting analyses of genomic DNA from a panel of independently derived multidrug-resistant cell lines identified mdr gene amplification in 10 of 12 cell lines studied. In individual cell lines showing gene amplification, the copy number of each of the three mdr genes was identical, suggesting that the three mdr genes became amplified as part of a single amplicon in these cells. Although increased expression of all three mdr genes was detected in 2 of 12 cell lines tested, multidrug resistance was associated in 10 of 12 lines with the independent overexpression of either mdr1 (7 of 12) or mdr3 (3 of 12) but not mdr2. mdr1 overexpression was consistently associated with gene amplification, while increased mdr3 expression was detected in certain cell lines that did not show gene amplification. Increased levels of mdr1 mRNA were linked to the overexpression of a P glycoprotein of apparent molecular weight 180,000 to 200,000, whereas increased mdr3 expression resulted in increased expression of a P glycoprotein of molecular weight 160,000 to 180,000. Our results suggest that at least two members of the mouse mdr gene family, mdr1 and mdr3, can independently confer multidrug resistance in the cell lines examined.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Characterization of the multidrug resistance protein expressed in cell clones stably transfected with the mouse mdr1 cDNA.

Structural features of the multidrug resistance protein encoded by the mouse mdr1 gene were studied in multidrug-resistant cell clones stably transfected with a biologically active cDNA clone. Independently derived transfectant cell clones, initially selected in Adriamycin, were shown to be cross-resistant to several drugs, including actinomycin D, amsacrine, mitoxantrone, VP-16, and vinblastine but remained sensitive to cis-platinum, 5-fluorouracil, arabinocytosine, and bleomycin. In drug-resistant transfectants the mdr1 gene product was greatly overexpressed as a polypeptide of apparent molecular weight 160,000-170,000. This protein was present in membrane enriched fractions and could be metabolically labeled with [3H )glucosamine, confirming that the transfected mdr1 gene encodes a membrane glycoprotein. The protein was found phosphorylated on serine residues and was shown to be photolabeled by both the calcium antagonist azidopine and the ATP analogue 8-azido ATP. Tryptic mapping of the ATP-photoaffinity labeled protein indicated that ATP crosslinking was site-specific and limited to two discrete peptide fragments of the protein, suggesting that the overexpressed mdr protein is capable of direct and specific ATP binding.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

A mouse analogue of the human carcinoembryonic antigen.

Functional human carcinoembryonic antigen (CEA)-like genes have been shown to be present in the mouse. Southern analyses of murine DNA using both human and murine CEA complementary DNA probes have revealed the presence of multiple CEA-like genes, while analyses of RNA from different mouse tissues showed CEA-like transcripts in adult colon and liver. Furthermore, a CEA-like protein, immunoprecipitable with a rabbit polyclonal serum raised against human CEA, has been detected in adult murine colon tissue. Several murine CEA complementary DNA clones have been isolated from a murine colon complementary DNA library, and characterization of one such clone demonstrates that both the N-terminal and the internal domains have been conserved between the two species. The existence of a murine counterpart of CEA strengthens the case for an essential function for this human tumor marker and provides an experimentally amenable system for elucidation of its biological properties.

Animals↗
PubMed1989

Mammalian multidrug-resistance gene: correlation of exon organization with structural domains and duplication of an ancestral gene.

Analysis of the nucleotide and deduced amino acid sequences of the biologically active mouse mdr1 cDNA clone indicates that the protein is formed by two highly homologous halves, each containing six putative transmembrane domains and a nucleotide-binding site. The duplicated unit shows high sequence homology to the proposed energy-coupling subunit of bacterial periplasmic transport proteins. We have cloned and characterized the mouse mdr1 gene and have analyzed the genomic organization of the two homologous halves forming the mdr1 protein. The gene spans 68 kilobases, is split into 28 exons, and the two homologous halves are encoded by 14 and 13 exons. The transcriptional initiation site of the gene has been mapped and putative TATA and consensus CAAT sequences have been found at positions -27 and -83, respectively. Discrete structural domains of the mdr1 protein are encoded by separate exons: Ten of the 12 putative transmembrane domains are encoded by individual exons and the two nucleotide-binding sites are each encoded by three exons. The exon/intron organization of the gene is conserved in the two highly homologous regions encoding the nucleotide-binding sites. The conservation of certain pairs of introns, together with the high degree of sequence homology, indicate that the mouse mdr1 gene originated from the duplication of an intron-containing ancestral gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Interactions between pesticide genes: model and experiment.

In response to years of intense selection pressure by organophosphate insecticides, several different insecticide resistance mechanisms have evolved in natural populations of the mosquito Culex pipiens. We examined interactions between two of the most important mechanisms using a four-compartment model of insecticide pharmacokinetics. The joint effect of different mechanisms of resistance can be expressed in terms of epistasis at the physiological level in this model. The type of epistasis predicted by the model depends on the particular physiological mechanisms of resistance involved. Resistance due to a reduced penetration of the insecticide combines multiplicatively with other resistance factors, but resistance due to detoxicative processes and to insensitivity of the target site combines additively. How the pattern of epistasis at the physiological level is translated into fitness epistasis in natural populations of this mosquito depends on the intensity and pattern of insecticide selection in the field.

Animals↗
PubMed1989

The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues.

The gene responsible for multidrug resistance (mdr), which encodes the P-glycoprotein, is a member of a multigene family. We have identified distinct mdr gene transcripts encoded by three separate mdr genes in the mouse. Expression levels of each mdr gene are dramatically different in various mouse tissues. Specific mdr RNA transcripts of approximately 4.5, 5, and 6 kilobases have been detected. Each of the mdr genes has a specific RNA transcript pattern. These results should be considered in relation to understanding the normal physiological function of the mdr multigene family.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Cloning and characterization of a second member of the mouse mdr gene family.

The mammalian mdr gene family comprises a small number of closely related genes. Previously, we have shown that one member, mdr1, has the capacity to convey multidrug resistance to drug-sensitive recipient cells in a gene transfer protocol. However, the functional characteristics of other members of this gene family have not been examined. In this report, we characterize a second member of the mdr gene family which we designated mdr2. We determined the nucleotide sequence corresponding to the complete coding region of this mdr2 transcript. The predicted amino acid sequence of this protein (1,276 amino acids) showed that it is a membrane glycoprotein highly homologous to mdr1 (85%), strongly suggesting that both genes originate from a common ancestor. Regions of divergence between mdr1 and mdr2 proteins are concentrated in two discrete segments of the predicted polypeptides, each approximately 100 residues in length. The mdr2 protein appears to be formed by the duplication of a structural unit which encodes three putative transmembrane loops and a predicted nucleotide-binding fold and is highly homologous to bacterial transport proteins such as hlyB. This strong homology suggests that mdr2 also participates in an energy-dependent membrane transport process. However, the direct relationship, if any, of this new member of the mdr family to multidrug resistance remains to be established. Knowledge of the complete nucleotide sequence and predicted amino acid sequence of the mdr2 gene product will enable the preparation of gene-specific probes and antibodies necessary to study the functional role of this gene in multidrug resistance and normal physiological processes.

Amino Acid Sequence↗
PubMed1987

Multiple incompatibilities within populations of Culex pipiens L. in southern France.

Strains of Culex pipiens derived from natural populations collected in southern France were crossed to determine their ability to give fertile offspring. Uni- and bi-directional incompatibilities occurred between these strains and each of them had its own crossing properties. Compatibility or incompatibility of a cross seemed to be related to the geographic distance separating the parental colonies, but not to their ecological origin (hypogeous or epigeous). Our results showed evidence for the variation in time of crossing properties of a strain.

Animals↗
PubMed1987

Treatment of severe platelet dysfunction and hemorrhage after cardiopulmonary bypass: reduction in blood product usage with desmopressin.

Impairment of platelet function commonly occurs after cardiopulmonary bypass, and may result in substantial bleeding. Because desmopressin acetate (a synthetic analogue of vasopressin) shortens bleeding time in a variety of platelet disorders, a controlled clinical trial of intravenous desmopressin was performed in 39 patients with excessive mediastinal bleeding (greater than 100 ml/h) and a prolonged template bleeding time (greater than 10 minutes) more than 2 hours after termination of cardiopulmonary bypass. Twenty-three desmopressin recipients and 16 control patients (no desmopressin) were similar in surgical procedure, pump time, platelet count, template bleeding time and amount of bleeding before therapy (p = NS). Compared with the control group, the patients receiving desmopressin (20 micrograms; mean 0.3 micrograms/kg) utilized fewer blood products (29 +/- 19 versus 15 +/- 13 units/patient; p less than 0.05), especially platelets (12 +/- 9 versus 4 +/- 7 units/patient; p = 0.004), while achieving a similarly effective reduction in mediastinal bleeding (4.8- and 4.3-fold, p = 0.001 for both). Severe platelet dysfunction was partially corrected within 1 hour after desmopressin infusion, during which interval no blood products were administered: the template bleeding time shortened (from 17 to 12.5 minutes, p less than 0.05), whereas the platelet count remained unchanged (at 96 +/- 35 and 105 +/- 31 X 10(3)/mm3, p = NS). The plasma levels of two factor VIII components increased: procoagulant activity (VIII:C) from 0.97 +/- 0.43 to 1.52 +/- 0.74 units/ml (p less than 0.05) and von Willebrand factor (VIII:vWF) from 1.28 to 1.78 units/ml (p less than 0.05); these increases correlated with the shortening of the bleeding time (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelet Disorders↗
PubMed1986

Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito.

An esterase gene from the mosquito Culex quinquefasciatus that is responsible for resistance to a variety of organophosphorus (OP) insecticides was cloned in lambda gt11 phage. This gene was used to investigate the genetic mechanism of the high production of the esterase B1 it encodes in OP-resistant Culex quinquefasciatus Say (Tem-R strain) from California. Adults of the Tem-R strain were found to possess at least 250 times more copies of the gene than adults of a susceptible strain (S-Lab). The finding that selection by pesticides may result in the amplification of genes encoding detoxifying enzymes in whole, normally developed, reproducing insects emphasizes the biological importance of this mechanism and opens new areas of investigation in pesticide resistance management.

Animals↗
PubMed1986

cDNA clones for liver cytochrome P-450s from individual aroclor-treated rats: constitutive expression of a new P-450 gene related to phenobarbital-inducible forms.

Differential hybridization and screening with cloned inserts was used to identify two families of cytochrome P-450 cDNA clones in libraries prepared from total liver poly(A)+RNA of individual Aroclor-treated rats. One family has cDNA inserts for the major phenobarbital-inducible P-450s, P-450b and P-450e. Two types of P-450e inserts were identified. In addition, irregular inserts were characterized from two clones (PB23 and PB24) of this group. The other family has cDNA inserts for the major 3-methylcholanthrene-inducible species, P-450c and P-450d. No coding sequence restriction site variants were detected among 26 P-450d and P-450c inserts analyzed. The restriction map of the irregular 2.2-kb PB23 insert has a P-450b-like portion, followed by a 3' extension that hybridizes to RNAs of 2.7 and 4.8 kb, which are also detectable with a classical P-450b probe. The PB23 insert and the 2.7- and 4.8-kb RNAs presumably represent 3' extensions of P-450b/P-450e mRNAs, polyadenylated at downstream sites. The 858-bp sequence of the PB24 insert encodes the carboxy-terminal portion of a P-450b/P-450e-like protein. There is approximately 20% divergence at the polypeptide level between the PB24 and P-450b/P-450e sequences; nevertheless, they share many essential features. A PB24-specific probe hybridizes to a 1.9-kb RNA species which is present in the liver of untreated rats and which is not appreciably induced by phenobarbital or Aroclor. The PB24 cDNA most likely represents a constitutive cytochrome P-450, related to phenobarbital-inducible forms.

Amino Acid Sequence↗