Biomedical subjects
Publications and source records attributed to P Gros.
Hemangioendotheliomas are vascular tumors of intermediate malignancy considered as low-grade angiosarcomas. The authors report two cases of a distinctive type, named retiform hemangioendothelioma because of its particular morphologic features, and recently delineated by Calonje and al. This tumor of the skin recurs frequently but has a very low metastatic rate.
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A case of angiosarcoma in the small bowel is reported. There were multifocal lesions in the duodeno-jejunum with peritoneal metastasis, revealed by an hemorrhagic syndrome. This tumor rare in the gastro-intestinal tract was epithelioïd. The authors present an histochemical and immunohistochemical study with a review of the literature.
BACKGROUND: Renal oncocytoma has several features that overlap with other renal neoplasms, including the eosinophilic subtype of chromophobe cell carcinoma. In fact, strict criteria for renal oncocytoma have not been well defined and remain a matter of controversy. Ultrastructural studies or sophisticated methods such as flow cytometry and cytogenetic techniques can be of great use in distinguishing the two tumors, but are difficult to propose as routine methods because of their limited availability. OBJECTIVE: To further characterize the histologic criteria of these tumors, we undertook a retrospective study to define the utility of routinely available histochemical and immunohistochemical techniques. DESIGN AND SETTING: Twenty-one cases of chromophobe cell carcinoma, eosinophilic subtype, and 103 cases of oncocytoma were tested with histochemical (Perls, periodic acid-Schiff, and Hale's colloidal iron) and immunohistochemical (peanut agglutinin antigen and UEA-1 for lectins; cytokeratin KL1, epithelial membrane antigen, vimentin, S100 protein, and lysozyme) staining. RESULTS: The antibodies tested and the histochemical staining using Hale's colloidal iron allowed eosinophilic chromophobe cell carcinoma to be distinguished by its characteristic reaction pattern. Seventy-six percent of the chromophobe cell carcinomas showed a microvacuolated pattern, and 89% of the renal oncocytomas showed an apical positivity with Hale's colloidal iron staining (P < .01). Peripheral cell accentuation reactivity for cytokeratin KL1 was observed in 66% of the chromophobe cell carcinoma cases, and apical cytoplasmic positivity was observed in 37% of the renal oncocytoma cases (P = .01). Significant patterns were observed with anti-epithelial membrane antigen and anti-peanut agglutinin antigen antibodies (P = .05 and P = .01, respectively). Positive reactions for vimentin, S100 protein, lysozyme, and UEA-1 were not significant characteristics. CONCLUSION: Our study demonstrated that a precise morphologic description associated with simple histochemical and immunohistochemical techniques provides sufficient criteria for a high level of discrimination between the eosinophilic subtype of chromophobe cell carcinoma and renal oncocytoma.
Dentigerous cysts, also called follicularor corono-dental cysts, are malformative odontogenic cysts in the WHO classification of odontogenic tumors. We review the general characteristics of these frequent odontogenic tumors which always occur in associated with an included tooth. Diagnosis is based on clinical features, X-ray findings and pathology results. We report the evolutive aspect in three patients of different ages.
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The mouse Nramp1 gene (Bcg/Ity/Lsh) controls innate defense to infection with intracellular parasites such as Mycobacterium, Salmonella, and Leishmania. Sequence analysis of Nramp1 predicts a hydrophobic, membrane-associated protein expressed exclusively in monocyte/macrophage lineages. A single G169D substitution within the fourth predicted transmembrane domain of Nramp1 is associated with susceptibility to infection (Bcg) in inbred mouse strains. To initiate the biochemical characterization of the Nramp1 protein and to analyze the molecular basis of susceptibility associated with the Nramp1(D169) allele, oligopeptides derived from Nramp1 and two fusion proteins containing the first 54 and the last 35 residues of Nramp1 were used to raise specific anti-Nramp1 antisera. In addition, a c-Myc epitope (EQKLISEEDL) was introduced in-frame at the C terminus of Nramp1 to follow its expression in a yeast heterologous system. Western analysis of crude membrane fractions from yeast demonstrated that Nramp1 is indeed an integral membrane protein (resistant to urea extraction). In resident Bcg(r) (129sv, Nramp1(G169)) macrophages, one of the anti-Nramp1 antisera identified by immunoprecipitation a protein of 90,000 to 100,000 apparent m.w. that was absent in macrophages from knockout mice bearing a null mutation at Nramp1. The Nramp1 protein could be metabolically labeled with orthophosphate and was sensitive to glycosylase treatment, verifying that Nramp1 is an integral membrane phosphoglycoprotein. Parallel analysis of macrophage extracts from Bcg(s) mouse strains (C57BL6/J and BALB/c, Nramp1(D169)) failed to detect mature Nrampl protein in these cells, suggesting that the G169D mutation at Nramp1 prevents proper maturation or membrane integration of the protein, resulting in its rapid degradation.
We have isolated and characterized cDNA clones encoding the human and rat Na,K-ATPase beta3 subunit isoform. The human cDNA encodes a polypeptide of 279 amino acids that exhibits primary sequence and secondary structure similarities to Na,K-ATPase beta subunit isoforms. Sequence comparisons showed that the human beta3 subunit closely resembles the beta3 subunit of Xenopus laevis (59% amino acid identity) and is less similar to the human Na,K-ATPase beta1 and beta2 subunits (38% and 48% amino acid identity, respectively). By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the beta3 subunit gene to murine chromosome 7. Northern blot analysis revealed that the beta3 subunit gene encodes two transcripts that are expressed in a variety of rat tissues including testis, brain, kidney, lung, stomach, small intestine, colon, spleen, and liver. Identification of the mammalian beta3 subunit suggests an even greater potential for Na,K-ATPase isoenzyme diversity than previously realized.
The exact role of the pfmdr1 gene in the emergence of drug resistance in the malarial parasite Plasmodium falciparum remains controversial. pfmdr1 is a member of the ATP binding cassette (ABC) superfamily of transporters that includes the mammalian P-glycoprotein family. We have introduced wild-type and mutant variants of the pfmdr1 gene in the yeast Saccharomyces cerevisiae and have analyzed the effect of pfmdr1 expression on cellular resistance to quinoline-containing antimalarial drugs. Yeast transformants expressing either wild-type or a mutant variant of mouse P-glycoprotein were also analyzed. Dose-response studies showed that expression of wild-type pfmdr1 causes cellular resistance to quinine, quinacrine, mefloquine, and halofantrine in yeast cells. Using quinacrine as substrate, we observed that increased resistance to this drug in pfmdr1 transformants was associated with decreased cellular accumulation and a concomitant increase in drug release from preloaded cells. The introduction of amino acid polymorphisms in TM11 of Pgh-1 (pfmdr1 product) associated with drug resistance in certain field isolates of P. falciparum abolished the capacity of this protein to confer drug resistance. Thus, these findings suggest that Pgh-1 may act as a drug transporter in a manner similar to mammalian P-glycoprotein and that sequence variants associated with drug-resistance pfmdr1 alleles behave as loss of function mutations.
We have used a total of 27 AXB/BXA recombinant inbred mouse strains to determine the chromosomal location of a newly identified gene encoding an alpha-subunit isoform of the sodium channel from Schwann cells, Scn9a. Linkage analysis established that Scn9a mapped to the proximal segment of mouse chromosome 2. The segregation of restriction fragment length polymorphisms in 145 progeny from a Mus spretus x C57BL/6J backcross indicates that Scn9a is very tightly linked to Scn1a (gene encoding the type I sodium channel alpha-subunit of the brain) and forms part of a cluster of four Scna genes located on mouse chromosome 2.
P-glycoprotein (P-gp) is an active transporter that can confer multidrug resistance by pumping cytotoxic drugs out of cells and tumors. P-gp is phosphorylated at several sites in the "linker" region, which separates the two halves of the molecule. To examine the role of phosphorylation in drug transport, we mutated P-gp such that it could no longer be phosphorylated by protein kinase C (PKC). When expressed in yeast, the ability of the mutant proteins to confer drug resistance, or to mediate [3H]vinblastine accumulation in secretory vesicles, was indistinguishable from that of wild type P-gp. A matched pair of mammalian cell lines were generated expressing wild type P-gp and a non-phosphorylatable mutant protein. Mutation of the phosphorylation sites did not alter P-gp expression or its subcellular localization. The transport properties of the mutant and wild type proteins were indistinguishable. Thus, phosphorylation of the linker of P-gp by PKC does not affect the rate of drug transport. In light of these data, the use of agents that alter PKC activity to reverse multidrug resistance in the clinic should be considered with caution.
P-glycoprotein (P-gp) is an integral membrane protein that causes multidrug resistance when overexpressed in tumor cells. Efforts to identify the position and polarity of its 12 putative transmembrane (TM) domains have so far failed to yield a consistent topological model. Recently, we have described a method for topology mapping based on the insertion of a small antigenic peptide epitope (YPYDVPDYA) in predicted intra- or extracellular loops of the protein. The tagged proteins are then functionally expressed in Chinese hamster ovary cells, and the polarity of the inserted tag with respect to plasma membrane is deduced by immunofluorescence in intact or permeabilized cells. We previously localized segments between TM1 and TM2, and TM5 and TM6 as extracellular and segments between TM2 and TM3 and downstream of TM6 as intracellular (Kast, C., Canfield, V., Levenson, R., and Gros, P. (1995) Biochemistry 34, 4402-4411). We have now inserted single epitope tags at positions 207, 235, 276, 741, 782, 797, 815, 849, 887, 961, and 1024; double epitope tags at positions 736, 849, and 961; and a triple epitope tag at position 849. Insertions of epitopes at positions 235, 736, 741, 849, 887, 961, and 1024 resulted in functional proteins, whereas insertions at positions 207, 276, 782, 797, and 815 abrogated the capacity of P-gp to confer multidrug resistance. The epitope tags inserted at positions 736, 849, and 961 were localized extracellularly, whereas tags at positions 235, 887, and 1024 mapped intracellularly. These results indicate that the intervening segments separated by TM4-TM5, TM10-TM11, and downstream of TM12 are cytoplasmic; segments delineated by TM7-TM8, TM9-TM10, and TM11-TM12 are extracellular. Our combined analysis of the amino- and carboxyl-terminal halves of P-gp supports a 12-TM domain topology with intracellular amino and carboxyl termini and ATP binding sites and an extracellular glycosylated loop (TM1-TM2) in agreement with hydropathy prediction. These results are clearly distinct from those obtained by the analysis of truncated P-gps in vitro and in heterologous expression systems.
The biochemical and genetic analyses of P-glycoprotein (P-gp) have indicated that the membrane-associated regions of P-gp play an important role in drug recognition and drug transport. Predicted transmembrane domain 11 (TM11) maps near a major drug binding site revealed by photoaffinity labeling, and mutations in this domain alter the substrate specificity of P-gp. To investigate further the role of TM11 in P-gp function in general, and substrate specificity in particular, each of the 21 residues of TM11 of the P-gp isoform encoded by the mouse mdr3 gene was independently mutated to alanine, or to glycine in the case of endogenous alanines. After transfection and overexpression in Chinese hamster ovary cells, pools of stable transfectants were analyzed for qualitative or quantitative deviations from the profile of resistance to vinblastine, adriamycin, colchicine, and actinomycin D displayed by the wild-type protein. While mutations at eight of the positions had no effect on P-gp function, 13 mutants showed a 2-10-fold reduction of activity against one of the four drugs tested. Although the phenotype of individual mutants was varied, replacements at most mutation-sensitive positions seemed to affect the drug resistance profiles rather than the overall activity of the mutant P-gp. When TM11 was projected in a alpha-helical configuration, the distribution of deleterious and neutral mutations was not random but segregated with a more hydrophobic (mutation-insensitive) face and a more hydrophilic (mutation-sensitive) face of a putative amphipathic helix. The alternate clustering pattern of deleterious vs neutral mutations in TM11 together with the altered drug resistance profile of deleterious mutants suggest that the more hydrophilic face of the TM11 helix may play an important structural or functional role in drug recognition and transport by P-gp. Finally, the conservation of the two residues most sensitive to mutations (Y949 and Y953) in TM11, and in the homologous TM5, of all mammalian P-gps and also in other ABC transporters, suggests that these residues and domains may play an important role in structural as well as mechanistic aspects common to this family of proteins.
The multidrug resistance-associated protein (MRP) is a member of the ATP binding cassette superfamily of transporters which includes the mammalian P-glycoproteins (P-gp) family. In order to facilitate the biochemical and genetic analyses of MRP, we have expressed human MRP in the yeast Saccharomyces cerevisiae and have compared its functional properties to those of the mouse Mdr3 P-gp isoform. Expression of both MRP and Mdr3 in the anthracycline hypersensitive mutant VASY2563 restored cellular resistance to Adriamycin in this mutant. MRP and Mdr3 expression produced pleiotropic effects on drug resistance in this mutant, as corresponding VASY2563 transformants also acquired resistance to the anti-fungal agent FK506 and to the K+/H+ ionophore valinomycin. The appearance of increased cellular resistance to the toxic effect of Adriamycin (ADM) in MRP and Mdr3 transformants was concomitant with a reduced intracellular accumulation of [14C]ADM in spheroplasts prepared from these cells. Moreover, MRP and Mdr3, but not control spheroplasts, could mediate a time-dependent reduction in the overall cell-associated [14C]ADM from preloaded cells, suggesting the presence of an active ADM transport mechanism in MRP and Mdr3 transformants. Finally, human MRP was found to complement the biological activity of the yeast peptide pheromone transporter Ste6 and partially restored mating in a sterile ste6 null mutant. These findings suggest that despite their relatively low level of structural homology, MRP and P-gp share similar functional aspects, since both proteins can mediate transport of chemotherapeutic drugs and the a mating peptide pheromone in yeast.
The Lps locus on mouse chromosome 4 controls host responsiveness to lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria. The C3H/HeJ inbred mouse strain is characterized by a mutant Lps allel (Lpsd) that renders it hyporesponsive to LPS and naturally tolerant of its lethal effects. To identify the Lps gene by a positional cloning strategy, we have generated a high-resolution linkage map of the chromosomal region surrounding this locus. We have analyzed a total of 1604 backcross mice from a preexisting interspecific backcross panel of 259 (Mus spretus x C57BL/6J)F1 x C57BL/6J and two novel panels of 597 (DBA/2J x C3H/HeJ)F1 x C3H/HeJ and 748 (C57BL/6J x C3H/HeJ)F1 x C3H/HeJ segregating at Lps. A total of 50 DNA markers have been mapped in a 11.8-cM span overlapping the Lps locus. This positions the Lps locus within a 1.1-cM interval, flanked proximally by a large cluster of markers, including three know genes (Cd301, Hxb, and Ambp), and distally by two microsatellite markers (D4Mit7/D4Mit178). The localization of the Lps locus is several centimorgans proximal to that previously assigned.
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