Characterization of two non-locus of enterocyte effacement-encoded type III-translocated effectors, NleC and NleD, in attaching and effacing pathogens

Intestinal colonization by enteropathogenic and enterohemorrhagic Escherichia coli requires the locus of enterocyte effacement-encoded type III secretion system. We report that NleC and NleD are translocated into host cells via this system. Deletion mutants induced attaching and effacing lesions in vitro, while infection of calves or lambs showed that neither gene was required for colonization.     

Cell division cycle control in embryonal and alveolar rhabdomyosarcomas

In this study, we investigated the mRNA level of several genes involved in cell cycle regulation in alveolar (ARMS) and embryonal rhabdomyosarcomas (ERMS). p21(Cip1), Cyclin D1, Cyclin D2, Cyclin D3, CDK2, and CDK4 were evaluated by RT-PCR. All (13 out of 13) ERMS expressed the p21(Cip1) gene compared with only 40% (4 out of 10) of the ARMS. Moreover, the amount of p21(Cip1) mRNA was noticeably higher in the ERMS samples than in the positive ARMS specimens. p27(Kip1) protein were analysed by immunohistochemical and immunoblotting. A noticeable difference was observed, in that ERMS had higher amounts of the cell cycle inhibitor compared with the ARMS. Finally, treatment of two rhabdomyosarcoma cell lines, RH-30 and RD, with butyrate, resulted in complete growth inhibition and in the upregulation of the p21(Cip1) and p27(Kip1) levels. Our results demonstrate that ERMS have a much higher level of p27(Kip1) and p21(Cip1) than the alveolar types, explaining, at least in part, the distinct features and outcomes (i.e. a poor prognosis of the alveolar type) of the two forms of this childhood solid cancer. Moreover, the data on butyrate-treated cell lines suggest that the two genes are potential novel therapeutic targets for the treatment of rhabdomyosarcomas.     

Membrane ruffling and invasion of human and avian cell lines is reduced for aflagellate mutants of Salmonella enterica serotype Enteritidis

Independent studies have demonstrated that flagella are associated with the invasive process of Salmonella enterica serotypes, and aflagellate derivatives of Salmonella enterica serotype Enteritidis are attenuated in murine and avian models of infection. One widely held view is that the motility afforded by flagella, probably aided by chemotactic responses, mediates the initial interaction between bacterium and host cell. The adherence and invasion properties of two S. Enteritidis wild-type strains and isogenic aflagellate mutants were assessed on HEp-2 and Div-1 cells that are of human and avian epithelial origin, respectively. Both aflagellate derivatives showed a significant reduction of invasion compared with wild type over the three hours of the assays. Complementation of the defective fliC allele recovered partially the wild-type phenotype. Examination of the bacterium-host cell interaction by electron and confocal microscopy approaches showed that wild-type bacteria induced ruffle formation and significant cytoskeletal rearrangements on HEp-2 cells within 5 minutes of contact. The aflagellate derivatives induced fewer ruffles than wild type. Ruffle formation on the Div-1 cell line was less pronounced than for HEp-2 cells for wild-type S. Enteritidis. Collectively, these data support the hypothesis that flagella play an active role in the early events of the invasive process.     

Differential DNA Methylation as a Tool for Noninvasive Prenatal Diagnosis (NIPD) of X Chromosome Aneuploidies

The demographic tendency in industrial countries to delay childbearing, coupled with the maternal age effect in common chromosomal aneuploidies and the risk to the fetus of invasive prenatal diagnosis, are potent drivers for the development of strategies for noninvasive prenatal diagnosis. One breakthrough has been the discovery of differentially methylated cell-free fetal DNA in the maternal circulation. We describe novel bisulfite conversion- and methylation-sensitive enzyme digestion DNA methylation-related approaches that we used to diagnose Turner syndrome from first trimester samples. We used an X-linked marker, EF3, and an autosomal marker, RASSF1A, to discriminate between placental and maternal blood cell DNA using real-time methylation-specific PCR after bisulfite conversion and real-time PCR after methylation-sensitive restriction digestion. By normalizing EF3 amplifications versus RASSF1A outputs, we were able to calculate sex chromosome/autosome ratios in chorionic villus samples, thus permitting us to correctly diagnose Turner syndrome. The identification of this new marker coupled with the strategy outlined here may be instrumental in the development of an efficient, noninvasive method of diagnosis of sex chromosome aneuploidies in plasma samples.Aneuploidies, ie, an abnormal number of chromosomes, are responsible for a range of genetic disorders. The most frequent aneuploidies compatible with life are represented by trisomy 21 causing Down syndrome, trisomy 13 causing Patau syndrome, trisomy 18 causing Edwards syndrome, and sex chromosome aneuploidies.Sex chromosome aneuploidies include 45,X causing Turner syndrome (1/2500 living females), 47,XXX associated with triple X syndrome (1/1000 live births), 47,XXY associated with Klinefelter syndrome (prevalence 1/500 live males) (for prevalence and incidence data refer to http://www.orpha.net/consor/cgi-bin/index.php, last accessed on March 9, 2010) and the 47,XYY karyotype (the incidence generally reported is 1/1000 live births). Compared to trisomy 21, 13, and 18, sex chromosome aneuploidies show less severe clinical phenotypes, but taken together, the incidence of sex chromosome aneuploidies is high and, in the case of 47,XXX, 47,XXY and 47,XYY, this is largely underestimated. Moreover, although the mortality in utero of fetuses with Turner syndrome is high, most fetuses with other sex chromosome aneuploidies survive to term.¹ Major malformations may occur in Turner syndrome, but not in the XXY Klinefelter, XXX and XYY syndromes.²To date, conventional prenatal diagnosis of genetic disorders has been based on the analysis of fetal cells obtained using invasive procedures such as amniocentesis and chorionic villus sampling (CVS). These techniques are very reliable, but the downside is that both are associated with a small but significant risk of fetal loss, ie, in the order of 0.5 to 1.0% of cases. For this reason, invasive prenatal diagnosis is offered only if the perceived risk of abnormal pregnancy, estimated by maternal age, ultrasonography and other noninvasive methods, exceeds the miscarriage risk.³Several groups have investigated noninvasive methods of prenatal diagnosis.³ Attempts have been made to isolate fetal nucleated cells from maternal blood^4,5,6 but their rarity and the possibility of cells persistent from previous pregnancies have so far made this strategy unreliable.Recent strategies for noninvasive prenatal diagnosis (NIPD) have been based on the observation in 1948 of the presence of cell free circulating nucleic acid in blood plasma⁷ and the increase in this plasma DNA in cancer.^8,9,10 More recently, Lo et al¹¹ demonstrated the presence of male fetal DNA in maternal plasma, by amplifying Y specific sequences. Moreover, it was found that cell free fetal DNA (cffDNA) in maternal plasma is fragmented¹² and the half-life is in the order of 16 minutes after delivery.¹³ The amount of cffDNA in maternal plasma DNA ranges between approximately 3 to 6% with a mean of 25.4 genome copies/ml of maternal plasma during early pregnancy.¹⁴ cffDNA from maternal plasma has been successfully used to determine fetal rhesus D (RhD) blood type,¹⁵ for determination of fetal sex,^14,16,17 thus limiting the need for invasive diagnosis in cases of sex specific pathologies as well as for the identification of some fetal disorders due to paternal genetic mutations or recessive conditions where parents are compound heterozygotes.^18,19 However, the presence of a great excess of free maternal DNA complicates the use of such methods.¹⁴The most important source of cffDNA released in maternal plasma during pregnancy appears to be the placenta,^20,21 whereas it has been suggested that the cell free maternal DNA (cfmDNA) originates from hematopoietic cells.²² On the basis of the placental origin of free fetal nucleic acids (cffDNA and cffRNA²³) and the finding of a chromosome 21 placenta-specific mRNA marker in maternal plasma,²⁴ an approach based on RNA-SNP allelic ratio has been reported, to detect aneuploidies of this chromosome.²⁴ Recently, Lo and co-workers²⁵ reported the use of digital PCR to determine the over-representation of chromosome 21 in trisomy 21 samples in mixtures of placental and maternal blood cell DNA, using samples containing at least 25% of fetal DNA, a concentration many fold higher than that present in a first trimester maternal plasma sample. A similar strategy, based on microfluidic digital PCR platform has been applied by Fan and co-workers²⁶ in the set-up of diagnosis of chromosome number abnormalities, on amniotic fluid and CVS samples.Recently, high throughput technologies, such as those based on parallel DNA-sequencing²⁷ are being applied to NIPD strategies. Read depth analysis was used to successfully identify chromosome 13, 18, and 21 aneuploidies of the fetus.²⁸ Chiu and co-workers²⁹ also applied high throughput DNA-sequencing to quantify the amount of unique chromosome 21 sequences from plasma (maternal and fetal), revealing a potential trisomy. However, high throughput DNA-sequencing is still costly and difficult to manage in a routine laboratory, due to the large bioinformatic and computer resources required for analysis. For the foreseeable future, it may be difficult to translate this approach for widespread noninvasive diagnosis of aneuploidies.Strategies exploiting differential DNA methylation may also be used to discriminate tissues/cells of different origin. DNA methylation, the major post-biosynthetic modification found throughout mammalian genomes, is involved in many important biological phenomena, such as X chromosome inactivation and genomic imprinting, and in controlling tissue-specific expression in adult somatic tissues.^30,31,32Differential DNA methylation between maternal and fetal DNA has been investigated for use in NIPD. Differential methylation between fetal CVS and maternal blood cell DNA was first reported in 2002³³ and three years later the first universal epigenetic marker of fetal DNA in maternal plasma, SERPINB5 was described. The promoter region of SERPINB5 is hypomethylated in placenta and hypermethylated in maternal blood cells. Fetal SERPINB5 was distinguished from maternal SERPINB5 in maternal plasma DNA³⁴ after bisulfite modification³⁵ and methylation specific PCR.³⁶ Subsequently, the application of bisulfite-independent methods were also reported.^37,38Despite the appeal of using epigenetic differences between maternal and fetal DNA to develop NIPD for aneuploidies, the search for chromosome specific markers is challenging: this is particularly true for X linked sequences. An alternative high-throughput approach for identifying chromosome specific methylated markers is based on immunoprecipitation of methylated DNA (MeDiP³⁹) coupled with high resolution tiling oligonucleotide array analysis. More than 2000 differentially methylated regions between placenta and maternal blood cells, on respectively chromosome 13, 18, 21, X and Y, both in non-genic regions and in the CpG islands have been recently reported using this approach.⁴⁰Taking advantage of these latter data, we selected three putative X-linked markers that are differentially methylated between placenta and maternal blood cell DNA, and after further validation we developed a NIPD strategy to determine copy number of fetal sex chromosomes.     

High frequency of homozygous deletions of CDK4I gene in childhood acute lymphoblastic leukaemia

Summary. To determine the incidence of homozygous deletions of the newly identified tumour suppressor gene, CDK4I, molecular genomic DNA analyses by PCR technique were performed on primary neoplastic cells from 22 childhood acute leukaemias obtained at presentation. The blast cells derived in all the analysed cases from bone marrow. We found that none of acute myeloblastic leukaemias (four cases) showed the CDK4I alteration, whereas 6/13 (46%) common acute lymphoblastic leukaemias (ALLs) displayed homozygous deletions. Moreover, and even more important, all the blasts purified from ALLs derived from early lymphoid precursors (three early-T ALLs and two pre-B ALLs) showed the absence of CDK4I gene. When the entire coding sequence of the CDK4I gene from samples without homozygous deletions was analysed by the single-strand conformational polymorphism method, no point mutations were identified. These results demonstrate that CDK4I gene deletions are very frequent and probably early events in childhood acute leukaemias of lymphoid origin and especially in early-T and pre-B ALLs. Moreover, the molecular mechanism of the loss of function of the gene is correlated, at least in childhood ALLs, almost exclusively to deletions and not to point mutations.     

Analysis of cyclin-dependent kinase inhibitor genes (CDKN2A,CDKN2B,and CDKN2C) in childhood rhabdomyosarcoma

p16^INK4A, p15^INK4B, and p18 proteins are highly specific inhibitors of cyclin-dependent serine/threonine kinase (CDK) activities required for G1-S transition in the eukaryotic cell division cycle. Mutations, mainly homozygous deletions, of the CDKN2A (p16^INK4A/MTSI) gene have been recently found in tumor cell lines and in many primary tumors. We looked for homozygous deletions of CDKN2A, CDKN2B (p15^INK4B), and CDKN2C (p18) in 12 primary rhabdomyosarcoma (RMS) specimens and in five cell lines established from this cancer type. By means of polymerase chain reaction (PCR) and PCR-single strand conformation polymorphism (PCR-SSCP), we analyzed the presence of biallelic gene deletion or point mutation causing gene function loss. All the examined tumor cell lines (100%) and three of 12 (25%) primary tumors showed homozygous deletion of CDKN2A. Furthermore, no aberrant bands in primary tumors were detected via SSCP, suggesting the absence of mutations in the coding region. In all cases the deleted area at 9p21 also involved the CDKN2B gene. Conversely, no homozygous deletion or point mutations were detected when CDKN2C was analyzed. Our results strongly indicate that the p16^INK4A (and/or p15^INK4B) protein plays a key role in the development and/or progression of childhood rhabdomyosarcoma and suggest that this CDK-inhibitor protein might control proliferation and/or differentiation of human muscle cells. Moreover, alteration of CDKN2C does not appear to be involved in the genesis of rhabdomyosarcoma. Genes Chromosom Cancer 15:217–222 (1996). © 1996 Wiley-Liss, Inc.     

Absence of methylthioadenosine phosphorylase in human gliomas

All normal mammalian tissues contain methylthioadenosine phosphorylase, which plays a role in the recycling of purines and methionine consumed during polyamine synthesis. A complete deficiency of methylthioadenosine phosphorylase has been reported in some human leukemias and lymphomas and in a few solid tumors. The exact incidence of the enzyme deficiency among fresh human tumor specimens has been difficult to establish because the measurement of enzyme catalytic activity is laborious and requires carefully preserved specimens. We have generated two antibodies against methylthioadenosine phosphorylase and have used them to develop a simple immunoblot assay for the enzyme. Specifically, studies showed that all cells with catalytically active methylthioadenosine phosphorylase had a 32-kDa band that reacted with the anti-enzyme antibodies. In a reciprocal manner, all malignant cell lines that were naturally deficient in methylthioadenosine phosphorylase activity lacked detectable immunoreactive enzyme protein. The immunoassay was used to analyze human gliomas. Seventy-five % (9 of 12) of the gliomas were completely methylthioadenosine phosphorylase deficient. This common metabolic difference between most gliomas and all normal cells is a potential target for tumor-specific chemotherapy.     

??-spectrinBari: a truncated ??-chain responsible for dominant hereditary spherocytosis

We describe a β-spectrin variant, named β-spectrin Bari, characterized by a truncated chain and associated with hereditary spherocytosis. The clinical phenotype consists of a moderately severe hemolytic anemia, splenomegaly, and spherocytes and acanthocytes in the blood smear. The occurrence of the truncated protein, that represents about 8% of the total β-spectrin occurring on the membrane, results in a marked spectrin deficiency. The altered protein is due to a single point mutation at position −2 (A->G) of the acceptor splice site of intron 16 leading to an aberrant β-spectrin message skipping exons 16 and 17 indistinguishable from that reported for β-spectrin Winston-Salem. We provide evidence that the mutated gene is transcribed but its mRNA is less abundant than either its normal counterpart or β-spectrin Winston-Salem mRNA. Our findings are an example of how mutations in different splice sites, although causing the same truncating effect, result in clearly different clinical pictures.