RESUMO
This study demonstrates, through a combination of stringent screening methods and thorough validation, that it is possible to identify transmembrane proteins preferentially expressed in primary breast tumour cells. mRNA was extracted from tumour cells isolated from invasive breast cancers and it was then subtracted against normal breast tissue mRNA prior to the generation of a signal sequence-trap library. Screening of the library identified 31 positive clones encoding 12 cell-surface and 12 secreted proteins. The expression of a subset of transmembrane genes was then interrogated using a high-throughput method (tissue microarray) coupled with cutting-edge in situ techniques in a large cohort of patients who had undergone uniform adjuvant chemotherapy. Expression of CD98 heavy chain (CD98HC) and low-level expression of the insulin-like growth factor 2 receptor/mannose-6-phosphate receptor (IGF2R/M6PR) correlated with poor patient prognosis in the whole cohort. Expression of bradykinin receptor B1 (BDKRB1) and testis enhanced gene transcript (TEGT) correlated with good prognosis in woman with oestrogen receptor (ER)-negative breast tumours. These results indicate that this combined approach of isolating primary tumour cells, generating a library to specifically isolate signal-sequence-containing transcripts, and in situ hybridization on tissue microarrays successfully identified novel prognostic markers (BDKRB1, CD98hc, and TEGT) and potential transmembrane therapeutic targets (CD98hc) in breast cancer.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Proteínas de Neoplasias/genética , Transcrição Gênica/genética , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/patologia , Feminino , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Hibridização In Situ/métodos , Molécula 1 de Adesão Intercelular/genética , Proteínas de Membrana/genética , Invasividade Neoplásica , Prognóstico , Proteínas/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Receptor B1 da Bradicinina/genética , Receptor IGF Tipo 2/genética , Receptores de Estrogênio/genética , Análise Serial de Tecidos/métodosRESUMO
The bloodsucking adult females of Phlebotomus perniciosus Newstead and P. longicuspis Nitzulescu (Diptera: Psychodidae) are important vectors of the protozoan Leishmania infantum Nicolle (Kinetoplastida: Trypanosomatidae) in western Mediterranean countries. The species status of the two phlebotomine sandflies was assessed, along with the epidemiological implications. Individual sandflies from three Moroccan Rif populations were characterized morphologically, isoenzymatically (by the isoelectrofocusing of alleles at the polymorphic enzyme loci of HK, GPI and PGM), and by comparative DNA sequence analysis of a fragment of mitochondrial Cytochrome b (mtDNA). By reference to the character profiles of specimens from other locations, including southern Spain and the type-locality countries, the Moroccan flies were placed in three lineages: first, the lineage of P. perniciosus, which contained two mtDNA sublineages, one (pnt) widely distributed and associated with the morphology of the male types from Malta, and the other (pna) associated with a P. longicuspis-like male morphology; second, the lineage of P. longicuspis sensu stricto, including typical forms from Tunisia; and third, a new sibling species of P. longicuspis. The mtDNA sublineage (pnt) of typical P. perniciosus was also found in some P. longicuspis from Morocco, indicating interspecific hybridization. The typical race of P. perniciosus occurs in Italy as well as in Malta, Tunisia and Morocco. It is replaced in southern Spain by the Iberian race (with the pni mtDNA sublineage). The discovery of interspecific gene introgression and a new sibling species mean that previous records of the two morphospecies do not necessarily reflect their true vectorial roles or geographical and ecological distributions.
Assuntos
DNA Mitocondrial/genética , Phlebotomus/enzimologia , Phlebotomus/genética , Alelos , Animais , Citocromos b/química , Citocromos b/genética , DNA Mitocondrial/química , Feminino , Genitália Masculina/ultraestrutura , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Focalização Isoelétrica , Isoenzimas , Masculino , Marrocos , Phlebotomus/anatomia & histologia , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Haplotypes of eight phlebotomine species were characterized by cycle sequencing a mitochondrial (mt) DNA fragment (cytochrome b to NADH1) amplified from single sandflies by PCR. Phlebotomus (Phlebotomus) papatasi displayed little variation throughout its large geographical range. We conclude that this vector of Leishmania major suffered a population bottleneck late in the Pleistocene and then radiated out from the eastern Mediterranean subregion. There was no support for a recent domestic lineage of P. papatasi. The mtDNA molecular clock in phlebotomines (subgenera Phlebotomus and Larroussius) was calibrated by reference to palaeogeographical events in Africa and the Mediterranean subregion. It fitted a pairwise nucleotide sequence divergence rate of 1.0-2.5% per million years. Co-evolution of L. major, its Phlebotomus vectors and mammalian reservoirs is discussed.
Assuntos
DNA Mitocondrial/genética , Haplótipos , Insetos Vetores/genética , Leishmania major , Phlebotomus/genética , Filogenia , África , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Molecular , Variação Genética/genética , Insetos Vetores/parasitologia , Região do Mediterrâneo , Dados de Sequência Molecular , Phlebotomus/parasitologia , Homologia de Sequência do Ácido NucleicoRESUMO
This study describes the preliminary applications of the squash blot technique in Tunisia, to detect Leishmania major in naturally infected Phlebotomus papatasi. 309 P. papatasi among 364 female sandflies squashed on to nylon Gene Screen DNA transfer membranes, were identified using the 3.2 kb ribosomal P. papatasi specific DNA probe described by Ready et al. A second hybridization using the Taq1 DNA probe described by Smith et al. (1989) allowed the detection and identification of the parasite in 15 (4.9%) of these P. papatasi specimens. The dissection of P. papatasi females during the same period and from the same biotopes showed an infection rate of 7.9% (9 positives among 113 dissected). The t proportion comparison test indicated that there is no significant statistical difference between the dissection and the squash blot technique for the estimation of infection rates of P. papatasi.
