Mitochondrial DNA (mtDNA) A 3243G mutation associated with an annular perimacular retinal atrophy

BACKGROUND: A point mutation at the locus 3243 of the mitonchondrial DNA (mtDNA) is associated with either the MIDD syndrome (maternally inherited diabetes, deafness), the MELAS syndrome (myopathy, encephalitis, lactic acidosis, stroke) or cardiac, digestive, endocrine or exocrine dysfunctions. We report a peculiar maculopathy in two patients with an mtDNA 3243 mutation. HISTORY AND SIGNS: Case 1: A visually asymptomatic 40-year-old woman was examined for screening of diabetic retinopathy. Visual acuity was 10 / 10 in both eyes. Case 2: A 54-year-old woman with deafness and diabetes complained of visual loss. Visual acuity was 6 / 10 for the right eye and 0.5 / 10 for the left eye. Both patients exhibited a chorioretinal areolar atrophy. Case 1 was followed over 15 years and exhibited a slow progression of the maculopathy with moderate loss of visual acuity to 6 / 10 in both eyes, but marked handicap from the annular scotoma. THERAPY AND OUTCOME: None. CONCLUSION: Both patients presented a perimacular annular retinal atrophy. Patients harbouring mtDNA 3243 mutation should be examined for the presence of a maculopathy, even if they are asymptomatic. Conversely, the finding of such a geographic maculopathy should suggest the possibility of a point mutation at the locus 3243 of the mitochondrial DNA, especially in the presences of diabetes mellitus and/or deafness

Transduction of CpG DNA-stimulated primary human B cells with bicistronic lentivectors.

Recently, using HIV-1-derived lentivectors, we obtained efficient transduction of primary human B lymphocytes cocultured with murine EL-4 B5 thymoma cells, but not of isolated B cells activated by CD40 ligation. Coculture with a cell line is problematic for gene therapy applications or study of gene functions. We have now found that transduction of B cells in a system using CpG DNA was comparable to that in the EL-4 B5 system. A monocistronic vector with a CMV promoter gave 32 +/- 4.7% green fluorescent protein (GFP)+ cells. A bicistronic vector, encoding IL-4 and GFP in the first and second cistrons, respectively, gave 14.2 +/- 2.1% GFP+ cells and IL-4 secretion of 1.3 +/- 0.2 ng/10(5) B cells/24 h. This was similar to results obtained in CD34+ cells using the elongation factor-1alpha promoter. Activated memory and naive B cells were transducible. After transduction with a bicistronic vector encoding a viral FLIP molecule, vFLIP was detectable by FACS or Western blot in GFP+, but not in GFP-, B cells, and 57% of sorted GFP+ B cells were protected against Fas ligand-induced cell death. This system should be useful for gene function research in primary B cells and development of gene therapies.

DNA fingerprinting of glioma cell lines and considerations on similarity measurements.

Glioma cell lines are an important tool for research in basic and translational neuro-oncology. Documentation of their genetic identity has become a requirement for scientific journals and grant applications to exclude cross-contamination and misidentification that lead to misinterpretation of results. Here, we report the standard 16 marker short tandem repeat (STR) DNA fingerprints for a panel of 39 widely used glioma cell lines as reference. Comparison of the fingerprints among themselves and with the large DSMZ database comprising 9 marker STRs for 2278 cell lines uncovered 3 misidentified cell lines and confirmed previously known cross-contaminations. Furthermore, 2 glioma cell lines exhibited identity scores of 0.8, which is proposed as the cutoff for detecting cross-contamination. Additional characteristics, comprising lack of a B-raf mutation in one line and a similarity score of 1 with the original tumor tissue in the other, excluded a cross-contamination. Subsequent simulation procedures suggested that, when using DNA fingerprints comprising only 9 STR markers, the commonly used similarity score of 0.8 is not sufficiently stringent to unambiguously differentiate the origin. DNA fingerprints are confounded by frequent genetic alterations in cancer cell lines, particularly loss of heterozygosity, that reduce the informativeness of STR markers and, thereby, the overall power for distinction. The similarity score depends on the number of markers measured; thus, more markers or additional cell line characteristics, such as information on specific mutations, may be necessary to clarify the origin.

Meta-analysis of estrogen response in MCF-7 distinguishes early target genes involved in signaling and cell proliferation from later target genes involved in cell cycle and DNA repair.

ABSTRACT: BACKGROUND: Many studies have been published outlining the global effects of 17 beta-estradiol (E2) on gene expression in human epithelial breast cancer derived MCF-7 cells. These studies show large variation in results, reporting between ~100 and ~1500 genes regulated by E2, with poor overlap. RESULTS: We performed a meta-analysis of these expression studies, using the Rank product method to obtain a more accurate and stable list of the differentially expressed genes, and of pathways regulated by E2. We analyzed 9 time-series data sets, concentrating on response at 3-4 hrs (early) and at 24 hrs (late). We found >1000 statistically significant probe sets after correction for multiple testing at 3-4 hrs, and >2000 significant probe sets at 24 hrs. Differentially expressed genes were examined by pathway analysis. This revealed 15 early response pathways, mostly related to cell signaling and proliferation, and 20 late response pathways, mostly related to breast cancer, cell division, DNA repair and recombination. CONCLUSIONS: Our results show that meta-analysis identified more differentially expressed genes than the individual studies, and that these genes act together in networks. These results provide new insight into E2 regulated mechanisms, especially in the context of breast cancer.

Activation of the mouse TATA-less and human TATA-containing UDP-glucuronosyltransferase 1A1 promoters by hepatocyte nuclear factor 1.

UDP-glucuronosyltransferase (UGT) 1A1 (UGT1A1) catalyzes the glucuronidation of bilirubin in liver. Among all UGT isoforms identified to date, it is the only relevant bilirubin-glucuronidating enzyme in human. Because glucuronoconjugation is the major route of bilirubin elimination, any genetic alteration that affects bilirubin glucuronosyltransferase activity may result in a more or less severe hyperbilirubinemia. In this study, we report the cloning and characterization of the transcriptional regulation of the mouse UGT1A1 gene. Primary-structure analysis of the mouse Thymidine Adevice promoter revealed marked differences with its human homolog. First, the mouse promoter lacks the highly polymorphic thymidine/adenine repeat occurring in the human promoter, which has been associated with some forms of hyperbilirubinemia. Second, an L1 transposon element, which is absent in the human promoter, is found 480 bp upstream of the transcription start site in mouse. Using the electromobility shift and DNase I footprinting experiments, we have identified a hepatocyte nuclear factor 1-binding site in the mouse UGT1A1 promoter that confers responsiveness to both factors HNF1alpha and HNF1beta in HEK293 cells. Furthermore, we show that this element, which is conserved in the human promoter, also confers strong HNF1 responsiveness to the human UGT1A1 gene. Together, these results provide evidence for a major regulatory function of this liver-enriched transcription factor in UGT1A1 activity in both rodents and human.

Directrius per a l'accessibilitat al contingut web, versió 1.0

Aquestes directrius expliquen com fer que el contingut web sigui accessible a persones amb discapacitats i s'adrecen a creadors de contingut (autors de pàgines web o dissenyadors de llocs web) i a creadors d'eines d'autor. L'objectiu principal d'aquestes directrius és promoure l'accessibilitat. Tanmateix, l'aplicació de les directrius facilitarà l'accés al contingut a tot tipus d'usuari, sigui quin sigui l'agent d'usuari usat (navegador web, navegador de veu, telèfon mòbil, ordinador de cotxe, etc.) o les condicions de l'entorn de consulta (entorns sorollosos, espais mal il·luminats, entorns en què no es poden usar les mans, etc.). L'aplicació d'aquestes directrius també ajudarà els usuaris a trobar la informació d'una manera més ràpida dins el web. Les directrius no pretenen desincentivar l'ús d'imatges, vídeo, etc., sinó que expliquen com fer que el contingut multimèdia sigui més accessible a una àmplia audiència.Aquest és un document de referència per a uns principis d'accessibilitat i idees de disseny. Algunes de les estratègies comentades tracten d'aspectes relatius a la internacionalització del web i a l'accés des de terminals mòbils. Tanmateix, el document se centra en l'accessibilitat i no tracta exhaustivament dels aspectes relacionats amb altres activitats del W3C. Si voleu més informació sobre aquests temes podeu consultar les pàgines inicials W3C Mobile Access Activity (per a l'accés des de terminals mòbils) i W3C Internationalization Activity (per als aspectes d'internacionalització).Aquest document està pensat per a ser estable en el temps i, per tant, no dóna informació específica sobre si els navegadors funcionen o no amb una determinada tecnologia, ja que aquesta informació varia molt ràpidament. Aquesta informació es pot trobar al web de la Web Accessibility Initiative ,WAI, (Iniciativa d'Accessibilitat Web) [WAI-UA-SUPPORT].Aquest document inclou un annex que organitza tots els punts de verificació ordenats per tema i per prioritat. Els punts de l'annex estan enllaçats a les respectives definicions en el document. Els temes recollits en l'annex inclouen les imatges, el contingut multimèdia, les taules, els marcs, els formularis i els scripts. L'annex es presenta en forma de taula o com a simple llista.Un document a part, amb el títol Techniques for Web Content Accessibility Guidelines 1.0 (Tècniques per a les directrius per a l'accessibilitat al contingut web, versió 1.0) ([TECHNIQUES]) explica com posar a la pràctica els punts citats fins aquí. El document de tècniques explica cada punt amb més detalls i dóna exemples usant el llenguatge d'etiquetatge d'hipertext (HTML), fulls d'estil en cascada (CSS), el llenguatge d'integració multimèdia sincronitzada (SMIL) o el llenguatge d'etiquetatge matemàtic (MathML). Aquest document també inclou tècniques per a provar o validar una pàgina web i un índex dels elements i atributs HTML amb les tècniques que els usen. El document de tècniques està pensat per a seguir de prop els canvis tecnològics i es preveu que s'actualitzi més sovint que les directrius.Nota: Algunes de les característiques descrites en les directrius no estan encara implementades en tots els navegadors o eines multimèdia; en concret pot ser que no es puguin utilitzar funcions noves d'HTML 4.0, de CSS1 o CSS2.Les Directrius per a l'accessibilitat al contingut web, versió 1.0 són part d'una col·lecció de directrius sobre accessibilitat publicades per la Web Accessibility Initiative, WAI (Iniciativa d'Accessibilitat Web). La col·lecció comprèn User Agent Accessibility Guidelines (Directrius d'accessibilitat per a agents d'usuari) [WAI-USERAGENT] i Authoring Tool Accessibility Guidelines (Directrius d'accessibilitat per a eines d'autor [WAI-AUTOOLS].

Identification of HIV integration sites in infected host genomic DNA.

The integration of the Human Immunodeficiency Virus (HIV) genetic information into the host genome is fundamental for its replication and long-term persistence in the host. Isolating and characterizing the integration sites can be useful for obtaining data such as identifying the specific genomic location of integration or understanding the forces dictating HIV integration site selection. The methods outlined in this article describe a highly efficient and precise technique for identifying HIV integration sites in the host genome on a small scale using molecular cloning techniques and standard sequencing or on a massive scale using 454 pyrosequencing.

Directrius per a l'accessibilitat al contingut web, versió 1.0. Recomanació de W3C, 5 de maig de 1999

Aquestes directrius expliquen com fer que el contingut web sigui accessible a persones ambdiscapacitats i s'adrecen a creadors de contingut (autors de pàgines web o dissenyadors de llocs web) ia creadors d'eines d'autor. L'objectiu principal d'aquestes directrius és promoure l'accessibilitat.Tanmateix, l'aplicació de les directrius facilitarà l'accés al contingut a tot tipus d'usuari, sigui quin siguil'agent d'usuari usat (navegador web, navegador de veu, telèfon mòbil, ordinador de cotxe, etc.) o les condicions de l'entorn de consulta (entorns sorollosos, espais mal il·luminats, entorns en què no es poden usar lesmans, etc.). L'aplicació d'aquestes directrius també ajudarà els usuaris a trobar la informació d'unamanera més ràpida dins el web. Les directrius no pretenen desincentivar l'ús d'imatges, vídeo, etc., sinóque expliquen com fer que el contingut multimèdia sigui més accessible a una àmplia audiència.Aquest és un document de referència per a uns principis d'accessibilitat i idees de disseny. Algunes deles estratègies comentades tracten d'aspectes relatius a la internacionalització del web i a l'accés desde terminals mòbils. Tanmateix, el document se centra en l'accessibilitat i no tracta exhaustivament delsaspectes relacionats amb altres activitats del W3C. Si voleu més informació sobre aquests temes podeuconsultar les pàgines inicials W3C Mobile Access Activity (per a l'accés des de terminals mòbils) i W3CInternationalization Activity (per als aspectes d'internacionalització). Aquest document està pensat per a ser estable en el temps i, per tant, no dóna informació específica sobre si els navegadors funcionen o no amb una determinada tecnologia, ja que aquesta informació varia molt ràpidament. Aquesta informació es pot trobar al web de la Web Accessibility Initiative ,WAI, (Iniciativa d'Accessibilitat Web) [WAI-UA-SUPPORT].Aquest document inclou un annex que organitza tots els punts de verificació ordenats per tema i perprioritat. Els punts de l'annex estan enllaçats a les respectives definicions en el document. Els temesrecollits en l'annex inclouen les imatges, el contingut multimèdia, les taules, els marcs, els formularis iels scripts. L'annex es presenta en forma de taula o com a simple llista. Un document a part, amb el títol Techniques for Web Content Accessibility Guidelines 1.0 (Tècniques per a les directrius per a l'accessibilitat al contingut web, versió 1.0) ([TECHNIQUES]) explica com posar a la pràctica els punts citats fins aquí. El document de tècniques explica cada punt amb més detalls i dóna exemples usant el llenguatge d'etiquetatge d'hipertext (HTML), fulls d'estil en cascada (CSS), el llenguatge d'integració multimèdia sincronitzada (SMIL) o el llenguatge d'etiquetatge matemàtic (MathML). Aquest document també inclou tècniques per a provar o validar una pàgina web i un índex Directrius per a l'accessibilitat al contingut web, versió 1.0 dels elements i atributs HTML amb les tècniques que els usen. El document de tècniques està pensat per a seguir de prop els canvis tecnològics i es preveu que s'actualitzi més sovint que les directrius.

Entry and transcription as key determinants of differences in CD4 T-cell permissiveness to human immunodeficiency virus type 1 infection.

Isolated primary human cells from different donors vary in their permissiveness-the ability of cells to be infected and sustain the replication of human immunodeficiency virus type 1 (HIV-1). We used replicating HIV-1 and single-cycle lentivirus vectors in a population approach to identify polymorphic steps during viral replication. We found that phytohemagglutinin-stimulated CD4(+) CD45RO(+) CD57(-) T cells from healthy blood donors (n = 128) exhibited a 5.2-log-unit range in virus production. For 20 selected donors representing the spectrum of CD4 T-cell permissiveness, we could attribute up to 42% of the total variance in virus production to entry factors and 48% to postentry steps. Efficacy at key intracellular steps of the replicative cycle (reverse transcription, integration, transcription and splicing, translation, and budding and release) varied from 0.71 to 1.45 log units among donors. However, interindividual differences in transcription efficiency alone accounted for 64 to 83% of the total variance in virus production that was attributable to postentry factors. While vesicular stomatitis virus G protein-mediated fusion was more efficacious than CCR5/CD4 entry, the latter resulted in greater transcriptional activity per proviral copy. The phenotype of provirus transcription was stable over time, indicating that it represents a genetic trait.

DNA mismatch repair gene MSH6 implicated in determining age at natural menopause.

The length of female reproductive lifespan is associated with multiple adverse outcomes, including breast cancer, cardiovascular disease and infertility. The biological processes that govern the timing of the beginning and end of reproductive life are not well understood. Genetic variants are known to contribute to ∼50% of the variation in both age at menarche and menopause, but to date the known genes explain <15% of the genetic component. We have used genome-wide association in a bivariate meta-analysis of both traits to identify genes involved in determining reproductive lifespan. We observed significant genetic correlation between the two traits using genome-wide complex trait analysis. However, we found no robust statistical evidence for individual variants with an effect on both traits. A novel association with age at menopause was detected for a variant rs1800932 in the mismatch repair gene MSH6 (P = 1.9 × 10(-9)), which was also associated with altered expression levels of MSH6 mRNA in multiple tissues. This study contributes to the growing evidence that DNA repair processes play a key role in ovarian ageing and could be an important therapeutic target for infertility.

Respiratory chain dysfunction associated with multiple mitochondrial DNA deletions in antiretroviral therapy-related lipodystrophy.

Highly-active antiretroviral therapy (HAART) can induce a characteristic lipodystrophy syndrome characterized by peripheral fat wasting and central adiposity, usually associated with hyperlipidaemia and insulin resistance [1,2]. Indirect data have led some authors to propose that mitochondrial dysfunction could play a role in this syndrome [3,4].To date, as recently outlined by Kakuda et al. [5] in this journal, HIV-infected patients developing lipodystrophy have not been studied for mitochondrial changes or respiratory chain capacity...

Activation of peroxisome proliferator-activated receptor beta/delta inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-kappaB activity via extracellular signal-related kinase 1/2.

OBJECTIVE: Chronic activation of the nuclear factor-kappaB (NF-kappaB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator-activated receptor (PPAR) beta/delta activation prevents inflammation in adipocytes. RESEARCH DESIGN AND METHODS AND RESULTS: First, we examined whether the PPARbeta/delta agonist GW501516 prevents lipopolysaccharide (LPS)-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the signal transducer and activator of transcription 3 (STAT3)-Suppressor of cytokine signaling 3 (SOCS3) pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-kappaB activation. Second, in in vivo studies, white adipose tissue from Zucker diabetic fatty (ZDF) rats, compared with that of lean rats, showed reduced PPARbeta/delta expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-kappaB DNA-binding activity. Furthermore, IL-6 expression and NF-kappaB DNA-binding activity was higher in white adipose tissue from PPARbeta/delta-null mice than in wild-type mice. Because mitogen-activated protein kinase-extracellular signal-related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-kappaB activation in adipocytes, we explored whether PPARbeta/delta prevented NF-kappaB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2 phosphorylation by LPS. Furthermore, white adipose tissue from animal showing constitutively increased NF-kappaB activity, such as ZDF rats and PPARbeta/delta-null mice, also showed enhanced phospho-ERK1/2 levels. CONCLUSIONS: These findings indicate that activation of PPARbeta/delta inhibits enhanced cytokine production in adipocytes by preventing NF-kappaB activation via ERK1/2, an effect that may help prevent insulin resistance.

Cloning of an interferon-gamma regulated human melanoma-associated antigen: identity to the intercellular adhesion molecule ICAM-1.

The human melanoma-associated antigen identified by the monoclonal antibody (mAb) Me14-D12 is a cell surface protein whose expression is induced by interferon-gamma (IFN-gamma). We have recently reported the molecular cloning of a genomic probe specific for the gene and mRNA of this protein. By screening with the genomic probe, we have now isolated a full length 3.0 kb cDNA from a Raji cell line-derived lambda-gt10 library. Sequence analysis of this cDNA showed a 99.8% homology with the intercellular adhesion molecule-1 (ICAM-1). Mouse Ltk- cells stably transfected with the human cDNA clone were found to express the ICAM-1 antigenic determinants detected by mAb Me14-D12 and a reference anti-ICAM-1 mAb, as judged by surface immunofluorescence. Immunoprecipitation of surface-iodinated proteins with mAb Me14-D12 revealed the presence of a 90 kD molecule with identical mobility to ICAM-1. In addition, mAb Me14-D12 could inhibit the phorbolester-stimulated aggregation of U937 cells. The findings show that the human melanoma-associated Me14-D12 antigen is the adhesion molecule ICAM-1.

Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogs.

BACKGROUND: Congenital, nonepidermolytic cornification disorders phenotypically resembling human autosomal recessive ichthyosis have been described in purebred dog breeds, including Jack Russell terrier (JRT) dogs. One cause of gene mutation important to humans and dogs is transposon insertions. OBJECTIVES: To describe an autosomal recessive, severe nonepidermolytic ichthyosis resembling lamellar ichthyosis (LI) in JRT dogs due to insertion of a long interspersed nucleotide element (LINE-1) in the transglutaminase 1 (TGM1) gene. METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Phenotypic information and genotyping with a canine microsatellite marker suggested TGM1 to be a candidate gene. Genomic DNA samples and cDNA generated from epidermal RNA were examined. Consequences of the mutation were evaluated by Western blotting, quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme activity from cultured keratinocytes. RESULTS: Affected dogs had generalized severe hyperkeratosis. Histological examination defined laminated to compact hyperkeratosis without epidermolysis; ultrastructurally, cornified envelopes were thin. Affected dogs were homozygous for a 1980-bp insertion within intron 9 of TGM1. The sequence of the insertion was that of a canine LINE-1 element. Quantitative RT-PCR indicated a significant decrease in TGM1 mRNA in affected dogs compared with wild-type. TGM1 protein was markedly decreased on immunoblotting, and membrane-associated enzyme activity was diminished in affected dogs. CONCLUSIONS: Based on morphological and molecular features, this disease is homologous with TGM1-deficient LI in humans, clinically models LI better than the genetically modified mouse and represents its first spontaneous animal model. This is the first reported form of LI due to transposon insertion.

Matrix metalloproteinase 9 (MMP-9/gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural killer cell-mediated cytotoxicity.

Shedding of intercellular adhesion molecule 1 (ICAM-1) is believed to play a role in tumor cell resistance to cell-mediated cytotoxicity. However, the mechanism whereby ICAM-1 is shed from the surface of tumor cells remains unclear. In this study, we have addressed the possibility that matrix metalloproteinases are implicated in ICAM-1 shedding. Our observations suggest a functional relationship between ICAM-1 and matrix metalloproteinase 9 (MMP-9) whereby ICAM-1 provides a cell surface docking mechanism for proMMP-9, which, upon activation, proteolytically cleaves the extracellular domain of ICAM-1 leading to its release from the cell surface. MMP-9-dependent shedding of ICAM-1 is found to augment tumor cell resistance to natural killer (NK) cell-mediated cytotoxicity. Taken together, our observations propose a mechanism for ICAM-1 shedding from the cell surface and provide support for MMP involvement in tumor cell evasion of immune surveillance.