Multifocal epithelial tumors and field cancerization from loss of mesenchymal CSL signaling.

It is currently unclear whether tissue changes surrounding multifocal epithelial tumors are a cause or consequence of cancer. Here, we provide evidence that loss of mesenchymal Notch/CSL signaling causes tissue alterations, including stromal atrophy and inflammation, which precede and are potent triggers for epithelial tumors. Mice carrying a mesenchymal-specific deletion of CSL/RBP-Jκ, a key Notch effector, exhibit spontaneous multifocal keratinocyte tumors that develop after dermal atrophy and inflammation. CSL-deficient dermal fibroblasts promote increased tumor cell proliferation through upregulation of c-Jun and c-Fos expression and consequently higher levels of diffusible growth factors, inflammatory cytokines, and matrix-remodeling enzymes. In human skin samples, stromal fields adjacent to multifocal premalignant actinic keratosis lesions exhibit decreased Notch/CSL signaling and associated molecular changes. Importantly, these changes in gene expression are also induced by UVA, a known environmental cause of cutaneous field cancerization and skin cancer.

The G0/G1 switch gene 2 is a novel PPAR target gene.

PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma are a group of transcription factors that are involved in numerous processes, including lipid metabolism and adipogenesis. By comparing liver mRNAs of wild-type and PPARalpha-null mice using microarrays, a novel putative target gene of PPARalpha, G0S2 (G0/G1 switch gene 2), was identified. Hepatic expression of G0S2 was up-regulated by fasting and by the PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. Surprisingly, the G0S2 mRNA level was highest in brown and white adipose tissue and was greatly up-regulated during mouse 3T3-L1 and human SGBS (Simpson-Golabi-Behmel syndrome) adipogenesis. Transactivation, gel shift and chromatin immunoprecipitation assays indicated that G0S2 is a direct PPARgamma and probable PPARalpha target gene with a functional PPRE (PPAR-responsive element) in its promoter. Up-regulation of G0S2 mRNA seemed to be specific for adipogenesis, and was not observed during osteogenesis or myogenesis. In 3T3-L1 fibroblasts, expression of G0S2 was associated with growth arrest, which is required for 3T3-L1 adipogenesis. Together, these data indicate that G0S2 is a novel target gene of PPARs that may be involved in adipocyte differentiation.

The 3' half of the mouse mammary tumor virus orf gene is not sufficient for its superantigen function in transgenic mice.

The Mouse Mammary Tumor Virus (MMTV) long terminal repeat contains an open reading frame (orf) of 960 nucleotides encoding a 36 kDa polypeptide with a putative transmembrane domain and five N-glycosylation sites in the N-terminal part of the protein. Transgenic mice bearing either the complete or the 3' terminal half of the orf sequence of MMTV-GR under the control of the SV40 promoter were raised. As shown previously by FACS analysis transgenic mice which express the complete orf gene have a significant deletion of V beta 14 expressing T cells at 6 weeks of age. Here we show that no clonal deletion of V beta 14 bearing T cells takes place in transgenic mice that contain orf sequences from the fifth ATG to the termination codon. The pattern of tissues expressing the truncated transgene was studied by the Polymerase Chain Reaction (PCR) and was very similar to the one obtained in the V beta 14 deleting animals. These data suggest that the amino-terminal portion of the ORF protein (pORF) is required for a superantigen function, while our previous data indicated that determinants from the carboxy-terminus play an important role for TCR V beta specificity.

Further delineation of the facial 13q14 deletion syndrome in 13 retinoblastoma patients.

Thirteen years ago, Motegi and colleagues (J Med Genet 1987;24:696-697) summarized the specific facial phenotype of six Japanese retinoblastoma patients with interstitial 13q14 deletions. Among a series of 228 propositi with retinoblastoma referred to the Lausanne Retinoblastoma Clinic for treatment and genetic counseling between 1986 and 1997, 13 (5.7%) were diagnosed with a cytogenetic de-novo 13q14 deletion. We confirm the presence of the reported facial phenotype in our population of Caucasian patients and describe additional clinical traits, thus extending the facial phenotype associated with the 13q14 deletion. Del(13q14) comprises, among others, cranial anomalies, frontal bossing, deeply grooved and long philtrum, depressed and broad nasal bridge, bulbous tip of the nose, thick lower lip, thin upper lip, broad cheeks, and large ears and lobules. Recognition of this particular facial appearance was instrumental in the genetic diagnosis of 13q deletions and in the presymptomatic diagnosis of retinoblastoma in a significant number of our cases. Identification of this phenotype in a retinoblastoma patient allows for efficient diagnosis of recurrence in his progeny and/or sibship, while its ignorance will compromise genetic counseling due to the possible difficulties in detecting large deletions by standard molecular mutation analysis. Recognition of this syndrome in newborns without known familial risk for retinoblastoma is even more important as it is a clear warning sign that indicates immediate ophthalmic examination.

Patient with Fanconi Syndrome (FS) and retinitis pigmentosa (RP) caused by a deletion and duplication of mitochondrial DNA (mtDNA).

BACKGROUND: We report a patient with a highly unusual presentation of a mitochondrial disorder. HISTORY AND SIGNS: An 8-year old girl presented with muscular cramps as well as height and weight deceleration. Investigations revealed lactic acidosis, electrolytic imbalance and urinary loss of glucose and electrolytes secondary to proximal renal tubulopathy consistent with Fanconi syndrome (FS). Ophthalmic examination revealed asymptomatic retinitis pigmentosa (RP) with no other ocular manifestations. A mitochondriopathy was suspected and genetic analysis performed. THERAPY AND OUTCOME: Southern blotting documented a heteroplasmic mutation of mtDNA with deletion/duplication. Three discrete mitochondrial genomes were detected: normal; deletion of 6.7 kb and a deletion/duplication consisting of 1 normal and 1 deleted genome. The relative proportions varied considerably between tissues. CONCLUSIONS: The association of FS and RP combines features of Kearns-Sayre syndrome and Pearson marrow-pancreas syndrome, without being typical of either. This highly unusual clinical presentation emphasises the need for systemic investigation of patients with FS and further underlines the importance of mtDNA analysis in patients with unexpected associations of affected tissues.

The role of the peptidyl prolyl isomerase Rrd1 in the transcriptional stress response

La régulation de la transcription est un processus complexe qui a évolué pendant des millions d’années permettant ainsi aux cellules de s’adapter aux changements environnementaux. Notre laboratoire étudie le rôle de la rapamycine, un agent immunosuppresseur et anticancéreux, qui mime la carence nutritionelle. Afin de comprendre les mécanismes impliqués dans la réponse a la rapamycine, nous recherchons des mutants de la levure Saccaromyces cerevisiae qui ont un phenotype altérée envers cette drogue. Nous avons identifié le gène RRD1, qui encode une peptidyl prolyl isomérase et dont la mutation rend les levures très résistantes à la rapamycine et il semble que se soit associé à une réponse transcriptionelle alterée. Mon projet de recherche de doctorat est d’identifier le rôle de Rrd1 dans la réponse à la rapamycine. Tout d’abord nous avons trouvé que Rrd1 interagit avec l’ARN polymérase II (RNAPII), plus spécifiquement avec son domaine C-terminal. En réponse à la rapamycine, Rrd1 induit un changement dans la conformation du domaine C-terminal in vivo permettant la régulation de l’association de RNAPII avec certains gènes. Des analyses in vitro ont également montré que cette action est directe et probablement liée à l’activité isomérase de Rrd1 suggérant un rôle pour Rrd1 dans la régulation de la transcription. Nous avons utilisé la technologie de ChIP sur micropuce pour localiser Rrd1 sur la majorité des gènes transcrits par RNAPII et montre que Rrd1 agit en tant que facteur d’élongation de RNAPII. Pour finir, des résultats suggèrent que Rrd1 n’est pas seulement impliqué dans la réponse à la rapamycine mais aussi à differents stress environnementaux, nous permettant ainsi d’établir que Rrd1 est un facteur d’élongation de la transcription requis pour la régulation de la transcription via RNAPII en réponse au stress.

Characterization of an escherichia coli elaC deletion mutant

The elaC gene of Escherichia coli encodes a binuclear zinc phosphodiesterase (ZiPD). ZiPD homologs from various species act as 3' tRNA processing endoribonucleases, and although the homologous gene in Bacillus subtilis is essential for viability [EMBO J. 22 (2003) 4534], the physiological function of E. coli ZiPD has remained enigmatic. In order to investigate the function of E. coli ZiPD we generated and characterized an E. coli elaC deletion mutant. Surprisingly, the E. coli elaC deletion mutant was viable and had wild-type like growth properties. Micro array-based transcriptional analysis indicated expression of the E. coli elaC gene at basal levels during aerobic growth. The elaC gene deletion had no effect on the expression of genes coding for RNases or amino-acyl tRNA synthetases or any other gene among a total of > 1300 genes probed. 2D-PAGE analysis showed that the elaC mutation, likewise, had no effect on the proteome. These results strengthen doubts about the involvement of E. coli ZiPD in tRNA maturation and suggest functional diversity within the ZiPD/ElaCl protein family. In addition to these unexpected features of the E. coli elaC deletion mutant, a sequence comparison of ZiPD (ElaCl) proteins revealed specific regions for either enterobacterial or mammalian ZiPD (ElaCl) proteins. (C) 2004 Elsevier Inc. All rights reserved.

Bradykinin receptor 1 activation exacerbates experimental focal and segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and alpha-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling. Kidney International (2011) 79, 1217-1227; doi:10.1038/ki.2011.14; published online 16 March 2011

EBNA-1 gene sequences in Brazilian and American patients with Hodgkin's disease

We examined the types of Epstein-Barr virus-associated nuclear antigen-1 (EBNA-1) gene carboxy (C)-terminal mutations occurring in Hodgkin's disease (HD) and reactive tissues from two different geographic regions. Previously reported EBNA-1 C-terminal region amino acid sequence variants, based on the amino acid at codon 487, include Prototype (P)-ala, which is found in the B95.8-derived prototype virus, P-thr, Variant (V)-leu, V-val, and V-pro. Using polymerase chain reaction (PCR) to amplify portions of the EBNA-1 gene, followed by DNA sequencing, we found a single EBNA-1 gene sequence variant in each tissue, whether reactive or neoplastic and whether from Brazil or the United States. Variant EBNA-1 gene sequences were more common in both neoplastic and non-neoplastic tissues from different geographic areas than the so-called prototype sequence. In the 17 Brazilian HD cases, 4 cases had P-thr variants and 13 had V-leu variants. In the six reactive tissues from Brazil, one had a P-ala variant, two had P-thr variants, and three had V-leu variants. In the 12 American HD cases, 2 had P-ala variants, 6 had P-thr variants, and 4 had V-leu variants. The 11 American reactive tissues included 2 P ala variants, 5 P-thr variants, and 4 V-leu variants. In both countries, there were similar variant EBNA-1 sequences present in normal tissues and HD cases. Compared with the P ala and P-thr cases, the V-leu cases were more likely to have the 30-bp latent membrane protein 1 (LMP1) gene deletion (P = 0.0075). In addition, cases of HD with the V-leu were statistically associated with a substitution of asparagine for glutamine at codon 322 of the C-terminal portion of the LMP1 gene. Our results suggest that any variation in EBNA-1 gene sequence is caused by a polymorphism present in pre-existing viral strains in the underlying population, and not a mutation occurring during oncogenesis. (C) 1999 by the American Society of Hematology.

The Effects of N Helix Deletion and Mutant F29W on the Ca2+ Binding and Functional Properties of Chicken Skeletal Muscle Troponin C

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L.B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (±Ca2+) of F29W and F29W/ NHdel and titration of the Ca2+-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca2+-activated enzymic activities. These effects are cumulative in the double mutant F29W/ NHdel. On the other hand, maximal isometric tension development in Ca2+-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.

Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counter-regulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages-cell types known to function in experimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS. Copyright © American Society for Investigative Pathology.

Detection of classical 17p11.2 deletions, an atypical deletion and RAI1 alterations in patients with features suggestive of Smith-Magenis syndrome

Smith-Magenis syndrome (SMS) is a complex disorder whose clinical features include mild to severe intellectual disability with speech delay, growth failure, brachycephaly, flat midface, short broad hands, and behavioral problems. SMS is typically caused by a large deletion on 17p11.2 that encompasses multiple genes including the retinoic acid induced 1, RAI1, gene or a mutation in the RAI1 gene. Here we have evaluated 30 patients with suspected SMS and identified SMS-associated classical 17p11.2 deletions in six patients, an atypical deletion of ∼139 kb that partially deletes the RAI1 gene in one patient, and RAI1 gene nonsynonymous alterations of unknown significance in two unrelated patients. The RAI1 mutant proteins showed no significant alterations in molecular weight, subcellular localization and transcriptional activity. Clinical features of patients with or without 17p11.2 deletions and mutations involving the RAI1 gene were compared to identify phenotypes that may be useful in diagnosing patients with SMS. © 2012 Macmillan Publishers Limited All rights reserved.

Diagnóstico molecular da alteração mutagênica nt 230 (del4) no gene MDR1 em cães

P-glycoprotein is an adenosine triphosphate (ATP)-driven drug efflux carrier responsible for transport of xenobiotics and multiple classes of drugs, many usually use in veterinary medicine. Encoded by MDR1 gene, also referred to as ABCB1, located on chromosome 14, is expressed in many tissues with secretory or excretory functions, such as liver, kidney and intestine, where it limits drug absorption from the gut and promotes drug excretion into the bile and urine of their substrates. In 2001, a 4 base pair gene deletion mutation in the canine MDR1 gene was identified as MDR1-1▲, ABCB1-1▲, MDR1 MDR1 nt 230 (del4) and associated with an non-functional Pglycoprotein. The clinical correlation is the (hyper) sensitivity of certain dogs breeds, mostly collies, to a few classes of drugs such as anticancer drugs (doxorubicin, vincristine, vinblastine), immunosuppressants (cyclosporine), antiparasitic drugs (ivermectin, moxidectin), steroids hormones (aldosterone, cortisol, dexamethasone), antimicrobial agents (tetracycline, doxycycline, levofloxacin, ketoconazole, itraconazole), analgesics (morphine, methadone), antidiarrheals (loperamide), antiepileptic agents (phenothiazine), cardiac drugs (digoxin, diltiazem, verapamil, talinolol) and others. Dogs with homozygous MDR1 nt 230 (del4) MDR1 mutations (MDR1 - / -) have a higher predisposition to intoxication with substrates of P-gp than heterozygous (MDR1 + / -) and these are more likely than dogs homozygous nonmutant (MDR1 +/ +). After the identification of nt230 (del4) mutation, several molecular techniques have been developed for identification of mutant animals as a diagnostic method. The importance of molecular diagnosis is, after the identification of mutant animals, establish treatment protocols safe, exclude this animals from reproduction (genetic selection program) and investigating the history of adverse drugs reactions... (Complete abstract click electronic access below)

Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.