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At least 253 records · Page 14Linked to original sources
PubMed1992

Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies.

Epstein-Barr virus (EBV), a human herpes virus with oncogenic potential, persists in B lymphoid tissues and is controlled by virus-specific cytotoxic T lymphocyte (CTL) surveillance. On reactivation in vitro, these CTLs recognize EBV-transformed lymphoblastoid cell lines (LCLs) in an HLA class I antigen-restricted fashion, but the viral antigens providing target epitopes for such recognition remain largely undefined. Here we have tested EBV-induced polyclonal CTL preparations from 16 virus-immune donors on appropriate fibroblast targets in which the eight EBV latent proteins normally found in LCLs (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, leader protein [LP], and latent membrane protein [LMP] 1 and 2) have been expressed individually from recombinant vaccinia virus vectors. Most donors gave multicomponent responses with two or more separate reactivities against different viral antigens. Although precise target antigen choice was clearly influenced by the donor's HLA class I type, a subset of latent proteins, namely EBNA 3A, 3B, and 3C, provided the dominant targets on a range of HLA backgrounds; thus, 15 of 16 donors gave CTL responses that contained reactivities to one or more proteins of this subset. Examples of responses to other latent proteins, namely LMP 2 and EBNA 2, were detected through specific HLA determinants, but we did not observe reactivities to EBNA 1, EBNA LP, or LMP 1. The bulk polyclonal CTL response in one donor, and components of that response in others, did not map to any of the known latent proteins, suggesting that other viral target antigens remain to be identified. This work has important implications for CTL control over EBV-positive malignancies where virus gene expression is often limited to specific subsets of latent proteins.

Antigens, Viral↗
PubMed1975

Prevention of folate deficiency by food fortification. IV. Identification of target groups in addition to pregnant women in an adult rural population.

In a rural Negro population subsisting on a predominantly maize meal diet, the incidence of folate deficiency was 43.8% in nonanaemic women in late pregnancy, 32.1% in nonpregnant women, and 18.6% in adult males. More than one-third of all subjects older than 60 were deficient. No instance of unequivocal vitamin B12 deficiency was revealed in 431 subjects sampled, and it is considered that the hazards of giving a small daily dose of folic acid in this population are negligible. These findings warrant food fortification with folic acid in this and similar population groups.

Adolescent↗
PubMed1995

Identification of target genes regulated by homeotic proteins in Drosophila melanogaster through genetic selection of Ultrabithorax protein-binding sites in yeast.

A method based on the transcriptional activation of a selectable reporter in yeast cells was used to identify genes regulated by the Ultrabithorax homeoproteins in Drosophila melanogaster. Fifty-three DNA fragments that can mediate activation by UBX isoform Ia in this test were recovered after screening 15% of the Drosophila genome. Half of these fragments represent single-copy sequences in the genome. Six single-copy fragments were investigated in detail, and each was found to reside near a transcription unit whose expression in the embryo is segmentally modulated as expected for targets of homoeotic genes. Four of these putative target genes are expressed in patterns that suggest roles in the development of regional specializations within mesoderm derivatives; in three cases these expression patterns depend on Ultrabithorax function. Extrapolation from this pilot study indicates that 85-170 candidate target genes can be identified by screening the entire Drosophila genome with UBX isoform Ia. With appropriate modifications, this approach should be applicable to other transcriptional regulators in diverse organisms.

Animals↗
PubMed1990

The response of human B cells to Mycobacterium leprae. Identification of target antigens following polyclonal activation in vitro.

We have investigated the B cell response to Mycobacterium leprae in leprosy patients and healthy controls. A comparison of Western-blotted proteins separated by two-dimensional gel electrophoresis and probed with pooled sera from LL and BT patients revealed distinct antigen recognition patterns for the two classifications of the disease. To characterize the circulating B cells capable of producing anti-M. leprae antibodies in vitro, peripheral blood lymphocyte cultures were activated polyclonally with an anti-CD3 mAb. The resulting culture supernatants were used to probe Western-blotted M.leprae proteins and contained antibody reactive with a 10 kd M.leprae antigen. This antibody was absent in stimulated culture supernatants from healthy occupational contacts or unexposed controls, suggesting the specificity of the response. Distinct repertoires of serum and culture supernatant anti-M.leprae antibodies were observed when Western-blotted antigens were probed after two-dimensional gel electrophoresis. This method for assay of specific antibody production against individual components present in a complex mixture of antigens after polyclonal activation in vitro may be used to study the regulation of B cell activation in leprosy and other diseases.

Adolescent↗
PubMed2005

Effect of a beta-lapachone-derived naphthoimidazole on Trypanosoma cruzi: identification of target organelles.

OBJECTIVES: Investigation of the mode of action of the naphthoimidazole N1, obtained from the reaction of beta-lapachone with benzaldehyde, which among 45 semi-synthetic derivatives of naphthoquinones isolated from Tabebuia sp. was one of the most active compounds against Trypanosoma cruzi trypomastigotes. METHODS: Quantification of the effect of N1 against the proliferative forms of T. cruzi, and investigation of potential targets in the parasite using electron microscopy and flow cytometry techniques. RESULTS: N1 presented the following order of activity: amastigotes > trypomastigotes > epimastigotes. The effect on intracellular forms was approximately 25 times higher than on macrophages and heart muscle cells. N1-treated parasites presented an abnormal chromatin condensation and mitochondrial damage. In epimastigotes, alterations of reservosomes were observed, and in trypomastigotes, a decrease in the electron density of acidocalcisomes was observed. In epimastigotes, the naphthoimidazole inhibited the activity of succinate cytochrome c reductase. Labelling with rhodamine 123 or Acridine Orange was decreased in both forms treated with N1. CONCLUSIONS: The results suggest that epimastigotes, reservosomes, mitochondrion, and nucleus contain N1 targets. In trypomastigotes, in which reservosomes are absent, the organelles affected by the compound were also the mitochondrion and nucleus, as well as acidocalcisomes, in which the decrease in electron density could be due to the use of polyphosphate as an alternative energy supply.

Acridine Orange↗
PubMed2005

Identification and targeted disruption of the gene encoding the main 3-ketosteroid dehydrogenase in Mycobacterium smegmatis.

The catabolic potential for sterol degradation of fast-growing mycobacteria is well known. However, no genes or enzymes responsible for the steroid degradation process have been identified as yet in these species. One of the key enzymes required for degradation of the steroid ring structure is 3-ketosteroid Delta(1)-dehydrogenase (KsdD). The recent annotation of the Mycobacterium smegmatis genome (TIGR database) revealed six KsdD homologues. Targeted disruption of the MSMEG5898 (ksdD-1) gene, but not the MSMEG4855 (ksdD-2) gene, resulted in partial inactivation of the cholesterol degradation pathway and accumulation of the intermediate 4-androstene-3,17-dione. This effect was reversible by the introduction of the wild-type ksdD-1 gene into M. smegmatis DeltaksdD-1 or overexpression of ksdD-2. The data indicate that KsdD1 is the main KsdD in M. smegmatis, but that KsdD2 is able to perform the cholesterol degradation process when overproduced.

Cloning, Molecular↗
PubMed1991

Identification of target genes of the homeotic gene Antennapedia by enhancer detection.

Localized expression of the homeotic gene Antennapedia (Antp) in Drosophila melanogaster is required for normal development of the thoracic segments. When the Antp gene is expressed ectopically in the larval primordium of the antenna, the antennal imaginal disc, the developmental fate of the disc is switched and the adult antenna is transformed to a mesothoracic leg. We screened approximately 550 different fly strains carrying single copies of an enhancer-detector transposon to identify regulatory elements and corresponding genes that are either activated or repressed in antennal discs in response to this transformation. Several regulatory elements that are either direct or indirect targets of Antp were found. One transposant that expresses the reporter gene (lacZ) in the antennal disc, but not in the leg disc, was studied in more detail. The enhancer detector in this strain is located near a similarly regulated gene at the spalt (sal) locus, which encodes a homeotic function involved in embryonic head and tail development. The expression of this newly discovered gene, spalt major (salm) is strongly repressed in gain-of-function mutants that express Antp in the antennal disc. Recessive loss-of-function mutations (Antp-) have the opposite developmental effect; they cause the differentiation of antennal structures in the second leg disc. Accordingly, salm is derepressed in clones of homozygous Antp- cells. Therefore, we conclude that Antp negatively regulates salm. The time course of the interaction and reporter gene fusion experiments suggests (but does not prove) a direct interaction between Antp and cis-regulatory elements of salm. Our analysis of several enhancer-detector strains suggests that the basic patterning information in the antennal and leg imaginal discs is very similar.

Alleles↗
PubMed1993

Identification and targeted gene disruption of cAR3, a cAMP receptor subtype expressed during multicellular stages of Dictyostelium development.

Extracellular cAMP acts through cell-surface receptors to coordinate the developmental program of Dictyostelium. A cAMP receptor (cAR1), which is expressed during early aggregation, has been cloned and sequenced previously. We have identified a new receptor subtype, cAR3, that has approximately 56% and 69% amino acid identity with cAR1 and cAR2, respectively. cAR1, cAR2, or cAR3 expressed from plasmid in growing Dictyostelium cells can be photoaffinity labeled with 8-N3[32P]cAMP and phosphorylated when stimulated with cAMP. cAR3 RNA was not present during growth but appeared during late aggregation. Its expression peaked at 9 hr and then fell to a reduced level that was maintained until culmination. The expression of cAR3 protein followed a similar pattern, but with a 3-hr lag, and reached a maximum at the mound stage. In contrast, cAR1 protein was expressed predominantly during early aggregation and at low levels during later stages. At their respective peaks of expression, there were approximately 5 x 10(3) cAR3 sites per cell compared with approximately 7 x 10(4) cAR2 sites per cell. The cAR3 gene was disrupted by homologous recombination in several different parental cell lines. Surprisingly, the car3- cell lines display no obvious phenotype.

Amino Acid Sequence↗
PubMed1999

Identification of target sites of the alpha2-Mcm1 repressor complex in the yeast genome.

The alpha2 and Mcm1 proteins bind DNA as a heterotetramer to repress transcription of cell-type-specific genes in the yeast Saccharomyces cerevisiae. Based on the DNA sequence requirements for binding by the alpha2-Mcm1 complex, we have searched the yeast genome for all potential alpha2-Mcm1 binding sites. Genes adjacent to the sites were examined for expression in the different cell mating types. These sites were further analyzed by cloning the sequences into a heterologous promoter and assaying for alpha2-Mcm1-dependent repression in vivo and DNA-binding affinity in vitro. Fifty-nine potential binding sites were identified in the search. Thirty-seven sites are located within or downstream of coding region of the gene. None of the sites assayed from this group are functional repressor sites in vivo or bound by the alpha2-Mcm1 complex in vitro. Among the remaining 22 sites, six are in the promoters of known alpha-specific genes and two other sites have an alpha2-Mcm1-dependent role in determining the direction of mating type switching. Among the remaining sequences, we have identified a functional site located in the promoter region of a previously uncharacterized gene, SCYJL170C. This site functions to repress transcription of a heterologous promoter and the alpha2-Mcm1 complex binds to the site in vitro. SCYJL170C is repressed by alpha2-Mcm1 in vivo and therefore using this method we have identified a new a-specific gene, which we call ASG7.

Binding Sites↗
PubMed2003

Global identification of target genes regulated by APETALA3 and PISTILLATA floral homeotic gene action.

Identifying the genes regulated by the floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI) is crucial for understanding the molecular mechanisms that lead to petal and stamen formation. We have used microarray analysis to conduct a broad survey of genes whose expression is affected by AP3 and PI activity. DNA microarrays consisting of 9216 Arabidopsis ESTs were screened with probes corresponding to mRNAs from different mutant and transgenic lines that misexpress AP3 and/or PI. The microarray results were further confirmed by RNA gel blot analyses. Our results suggest that AP3 and PI regulate a relatively small number of genes, implying that many genes used in petal and stamen development are not tissue specific and likely have roles in other processes as well. We recovered genes similar to previously identified petal- and stamen-expressed genes as well as genes that were not implicated previously in petal and stamen development. A very low percentage of the genes recovered encoded transcription factors. This finding suggests that AP3 and PI act relatively directly to regulate the genes required for the basic cellular processes responsible for petal and stamen morphogenesis.

Arabidopsis↗
PubMed2000

Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins.

The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.

Amino Acid Sequence↗
PubMed2004

On the way to targeted therapy of mast cell neoplasms: identification of molecular targets in neoplastic mast cells and evaluation of arising treatment concepts.

Several emerging treatment concepts for myeloid neoplasms are based on novel drugs targeting cell surface antigens, signalling pathways, or critical effector molecules. Systemic mastocytosis is a haematopoietic neoplasm that behaves as an indolent myeloproliferative disease in most patients, but can also present as aggressive disease or even as an acute leukaemia. In patients with aggressive disease or mast cell leukaemia, the response to conventional therapy is poor in most cases, and the prognosis is grave. Therefore, a number of attempts have been made to define novel treatment strategies for these patients. One promising approach may be to identify novel targets and to develop targeted drug therapies. In this article, we support the notion that neoplastic mast cells indeed express a number of potential molecular targets including immunoreactive CD antigens, the microphthalmia transcription factor (MITF), and members of the Bcl-2 family. In addition, the tyrosine kinase receptor KIT and downstream signalling pathways have been proposed as targets of a specific pharmacological intervention. A particular challenge is the disease-related D816V-mutated variant of KIT, which is resistant against diverse tyrosine kinase inhibitors including STI571, but may be sensitive to more recently developed targeted compounds. The therapeutic potential of target-specific approaches in malignant mast cell disorders should be evaluated in forthcoming clinical trials in the near future.

Antigens, CD↗
PubMed1997

Role of Escherichia coli histone-like nucleoid-structuring protein in bacterial metabolism and stress response--identification of targets by two-dimensional electrophoresis.

The histone-like nucleoid-structuring protein, H-NS, is a major bacterial chromatin component which influences DNA structure and gene expression. Mutations in hns, the structural gene of H-NS protein, have been shown to result in highly pleiotropic effects in Escherichia coli cells. In this study, we have initiated an index of the proteins whose synthesis is, directly or indirectly regulated by H-NS. Using two-dimensional gel electrophoresis, we have examined the global changes in gene expression which occured in an hns background compared with its wild-type parent. In addition, we analysed the effects of mutations in two other genes i.e. lrp and pta, which are also involved in global regulatory pathways. Although these comparative analyses revealed several common differences, thus suggesting possible interactions between these regulatory mechanisms, i.e. H-NS, Lrp (leucine-responsive regulatory protein) and acetylphosphate, the most extensive modifications occurred in an hns mutant. Among the polypeptides whose level of synthesis was specifically altered in an hns mutant, several corresponded to H-NS targets previously identified by classical selection methods. Moreover, the present study allows us to characterize several H-NS targets, which were identified either by comparison with the E. coli two-dimensional reference maps or by microsequencing procedure. Many of these newly identified polypeptides are involved in adaptation of E. coli cells to environmental challenges, and one of them could be involved in bacterial virulence. Finally, synthesis of several proteins belonging to the heat-shock regulon, more particularly molecular chaperones, was induced in an hns mutant.

Amino Acid Sequence↗
PubMed1999

Identification of target genes of oncogenic transcription factors.

Disregulation of many transcription factors is associated with the development of human neoplasia. Transcription factors regulate cell growth, differentiation, and apoptosis by binding to specific DNA sequences within the promoter regions of growth-regulatory genes and modulating expression of these genes. This simple model is complicated by the fact that mammalian transcription factors are often members of large protein families that bind to similar DNA sequences. This raises the question as to whether members of a particular family regulate expression of overlapping or unique sets of genes. This review is focused on addressing this question using the Ets, Myc, and E2F transcription factor families as examples. Deregulated activity of some, but not all, members of these families is observed in cancer. Here, we summarize the data illustrating the concept that binding of individual members of these families of factors can result in promoter-specific responses and review the studies that have provided some insight into how target gene specificity is achieved. Since, for all of these oncogenic transcription factors, it remains unclear exactly which target genes are important in neoplasia, we have also reviewed the many approaches researchers are using to identify target genes of the various Ets, Myc, and E2F family members.

Carrier Proteins↗
PubMed1998

Catheter ablation of ventricular tachycardia in patients with right ventricular dysplasia: Identification of target sites by entrainment mapping techniques.

OBJECTIVE: To identify target sites for radiofrequency ablation of ventricular tachycardia (VT) by entrainment mapping techniques in patients with arrhythmogenic right ventricular dysplasia. METHODS: Entrainment mapping and radiofrequency ablation of eight VTs was performed in seven patients. Radiofrequency ablation was applied at 31 reentry circuits sites that were classified based on findings during entrainment. RESULTS: By entrainment criteria the 31 sites were classified as: exit sites (n = 12), proximal sites (n = 6), and outer loop sites (n = 13). Radiofrequency current application terminated VT at 7 of 31 sites: 2 of 12 exit sites (17%), 4 of 6 proximal sites (67%), and 1 of 13 outer loop sites (8%). CONCLUSION: Radiofrequency ablation terminated VTs most often at sites proximal to the exit as opposed to outer loop sites and exit sites (P = 0.05). The critical isthmus for ablation of VT in right ventricular dysplasia often may be distant to the exit.

Arrhythmogenic Right Ventricular Dysplasia↗
PubMed2001

Specific identification and targeted characterization of Bifidobacterium lactis from different environmental isolates by a combined multiplex-PCR approach.

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.

Bacterial Typing Techniques↗