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At least 1,135 records · Page 63Linked to original sources
PubMed1996

Microsatellite instability in sporadic human breast cancers.

Human breast-cancer specimens from 100 patients were analyzed for microsatellite instability (referred to as replication error; RER) at 12 genomic loci on 7 chromosomes, and results were correlated with clinicopathologic characteristics. In 42 of 100 breast-cancer patients, we investigated whether RER was associated with the amplification of oncogenes and/or suppression of tumor-suppressor genes. Of the 100 patients, 8 (8%) were RER-positive at one or more chromosomal loci. The majority of RER-positive patients had early-stage disease with ER-positive tumors, suggesting that RER occurs early in breast tumorigenesis. However, no significant correlation was observed between RER and oncogenes or tumor-suppressor genes. Thus, the mechanism of RER in sporadic human breast cancer may be independent of the multi-step carcinogenesis caused by the alterations of oncogenes and tumor-suppressor genes.

Breast Neoplasms↗
PubMed1996

cDNA expression and human two-dimensional gel protein databases: towards integrating DNA and protein information.

The rapid progress in characterizing genes and mRNAs (expressed sequence tags, ESTs) as a result of the Human Genome Project makes it imperative to develop strategies to interface DNA mapping and sequencing data with protein information, as the latter orchestrate most cellular functions. Presently, the only technique able to resolve and record the thousands of proteins present in cells and tissues is two-dimensional (2-D) gel electrophoresis in combination with computer-aided technology to scan the gels, make synthetic images, assign numbers to individual spots as well as to enter qualitative and quantitative information. To date, comprehensive 2-D gel databases containing information about various properties of proteins (cellular localization, identification, regulatory properties, partial amino acid sequences, etc.) have been established (available on the internet: http:@biobase.dk/cgi-bin/celis). What remains is to provide a link between these data and the forthcoming information from the Human Genome Project. We are pursuing two approaches to achieve this goal: (i) microsequencing and mass spectrometry analysis of proteins resolved from 2-D gels and (ii) expression of cDNAs in the vaccinia virus expression system. Using the latter approach we have expressed about 60 cDNAs in human cells under conditions that faithfully reproduce post-translational trimmings and modifications of the proteins. The method, in combination with 2-D gel electrophoresis, allows precise matching of almost any cDNA to its protein product, irrespective of the protein abundance.

Animals↗
PubMedDate not supplied

Genetic retargeting of adenovirus vectors: functionality of targeting ligands and their influence on virus viability.

BACKGROUND: We studied the ability of adenovirus type 5 (Ad5) to encapsidate new cellular ligands carried by their fibers to yield functional retargeted vectors for gene therapy. Recombinant Ad5 fibers containing shaft repeats 1 to 7 and an extrinsic trimerization motif, and terminated by its native knob or amino acid motifs containing RGD, have been rescued into infectious virions. METHODS: Polypeptide ligands of cell surface molecules, including single-chain antibodies or epidermal growth factor, were cloned into recombinant fibers. Phenotypic analysis of fiber constructs and rescuing into the Ad5 genome were performed. Recombinant viruses were characterized with reference to fiber content, growth rate and infectivity. RESULTS: A major limiting factor for recovering viable recombinant Ad5 carrying fiber-fused polypeptide ligands was apparently the ability of the ligand to fold correctly within the cellular cytoplasm. This constraint has previously not been systematically evaluated in the literature. Phenotypic analysis of the fiber-ligand fusions showed that their degree of cytoplasmic solubility correlated with their ability to yield viable Ad5 vectors. Our results suggested that the fiber manipulations diminish virus growth rate, probably through different, opposing effects: (i) the reduced shaft length increases fiber solubility in the absence of the knob but (ii) diminishes virus entry, and (iii) the absence of the knob alters the overall protein composition of the virion and decreases its fiber copy number. CONCLUSIONS: Based on our findings, cytoplasmic solubility and cytoplasmic ligand reactivity of fiber-ligand fusion proteins are the best prediction criterion for viability and recovery of genetically retargeted Ad vectors.

Adenoviridae↗
PubMed2006

Relationship between classic Hodgkin lymphoma and overlapping large cell lymphoma investigated by comparative expressed sequence hybridization expression profiling.

There is a diagnostic grey zone between classic Hodgkin lymphoma (cHL) and some non-Hodgkin lymphoma (NHL), including primary mediastinal B cell lymphoma, diffuse large B cell lymphoma, and anaplastic large cell lymphoma. They all have some morphological and/or phenotypic features in common. To investigate this, we undertook an expression profiling study of these lymphomas using comparative expressed sequence hybridization. This technique detects chromosomal regions that are differentially expressed between a test and a reference tissue in a manner similar to comparative genomic hybridization, and is particularly suitable when the number of informative biopsies is limited. Using this approach, we identified a unique expression profile for all lymphoma types investigated. Unsupervised hierarchical cluster analysis of the acquired data showed that cHL separates from all investigated NHLs, including ALCL-like HL. Moreover, anaplastic lymphoma kinase (ALK)-negative ALCL clustered in a separate branch together with ALCL-like HL. Thus, analysing the neoplastic cells concurrently with their microenvironment, ALK-negative ALCL and ALCL-like HL seem to be related to each other, while cHL constitutes a separate lymphoma entity.

Adolescent↗
PubMed1993

The CEPH consortium linkage map of human chromosome 13.

The CEPH consortium map of chromosome 13 is presented. This map contains 59 loci defined by genotypes generated from CEPH family DNAs with 94 different probe and restriction enzyme combinations contributed by 9 laboratories. A total of 25 loci have been placed on the map with likelihood support of at least 1000:1. The map extends from loci in the centromeric region of chromosome 13 to the terminal band of the long arm. Multipoint linkage analyses provided estimates that the male, female, and sex-averaged maps extend for 158, 203, and 178 cM respectively. The largest interval is 24 cM and is between D13Z1 (alpha RI) and ATP1AL1. The mean genetic distance between the 25 uniquely placed loci is 7 cM.

Chromosome Mapping↗
PubMed2005

Synthesis of complex carbohydrates and glyconjugates: enzymatic synthesis of globotetraose using alpha-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus infuenzae strain Rd.

The lipopolysaccharide of capsule-deficient Haemophilus infuenzae strain Rd contains an N-acetylgalactosamine residue attached to the terminal globotriose moiety in the Hex5 glycoform. Genome analysis identified an open reading frame, HI1578, referred to as LgtD, whose amino acid sequence shows a significant level of similarity to those of a number of bacterial glycosyltransferases involved in lipopolysaccharide biosynthesis. To investigate its function, overexpression and biochemical characterization were performed. Most of the protein was obtained in a highly soluble and active form. Standard glycosyltransferase assay, high-performance liquid chromatography (HPLC), and liquid chromatography (LC)/mass spectrometry (MS) show that LgtD is an N-acetylgalactosaminyltransferase with high donor substrate specificity, and globotriose is a highly preferred acceptor substrate for the enzyme.

Amino Acid Sequence↗
PubMed1993

Genomic damage and its repair in young and aging brain.

A brief review of the available information concerning age-related genomic (DNA) damage and its repair, with special reference to brain tissue, is presented. The usefulness of examining the validity of DNA-damage and repair hypothesis of aging in a postmitotic cell like neuron is emphasized. The limited number of reports that exist on brain seem to overwhelmingly support the accumulation of DNA damage with age. However, results regarding the age-dependent decline in DNA-repair capacity are conflicting and divided. The possible reasons for these discrepancies are discussed in light of the gathering evidence, including some human genetic disorders, to indicate how complex is the DNA-repair system in higher animals. It is suggested that assessment of repair potential of neurons with respect to a specific damage in a specific gene might yield more definitive answers about the DNA-repair process and its role in aging.

Aging↗
PubMed2006

Genetic analysis and fine mapping of a rice brown planthopper (Nilaparvata lugens Stål) resistance gene bph19(t).

Genetic analysis and fine mapping of a resistance gene against brown planthopper (BPH) biotype 2 in rice was performed using two F(2) populations derived from two crosses between a resistant indica cultivar (cv.), AS20-1, and two susceptible japonica cvs., Aichi Asahi and Lijiangxintuanheigu. Insect resistance was evaluated using F(1) plants and the two F(2) populations. The results showed that a single recessive gene, tentatively designated as bph19(t), conditioned the resistance in AS20-1. A linkage analysis, mainly employing microsatellite markers, was carried out in the two F(2) populations through bulked segregant analysis and recessive class analysis (RCA), in combination with bioinformatics analysis (BIA). The resistance gene locus bph19(t) was finely mapped to a region of about 1.0 cM on the short arm of chromosome 3, flanked by markers RM6308 and RM3134, where one known marker RM1022, and four new markers, b1, b2, b3 and b4, developed in the present study were co-segregating with the locus. To physically map this locus, the bph19(t)-linked markers were landed on bacterial artificial chromosome or P1 artificial chromosome clones of the reference cv., Nipponbare, released by the International Rice Genome Sequencing Project. Sequence information of these clones was used to construct a physical map of the bph19(t) locus, in silico, by BIA. The bph19(t) locus was physically defined to an interval of about 60 kb. The detailed genetic and physical maps of the bph19(t) locus will facilitate marker-assisted gene pyramiding and cloning.

Animals↗
PubMed2001

Mapping and sequencing rat dishevelled-1: a candidate gene for cerebral ischaemic insult in a rat model of stroke.

A quantitative trait locus on chromosome 5 in the rat is linked to sensitivity to brain ischemia in the stroke-prone spontaneously hypertensive rat (SHRSP). The genes encoding atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) that map to this location have been excluded as candidate genes. We examined dishevelled-1 (DVL-1) as a further candidate gene. DVL-1 had not yet been identified in the rat, but Anp, Bnp, and DVL-1 map to the homologous regions of the rat chromosome 5 quantitative trait locus in both mice and man. Furthermore, DVL-1 is involved in the Notch signalling system, which plays a role in the disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, the symptoms of, which include ischaemic stroke. We show with radiation hybrid mapping that rat DVL-1 indeed maps to chromosome 5, where it is positioned immediately next to microsatellite marker D5Rat49. We sequenced the complete coding sequence and a large part of the intronic genomic sequence for the SHRSP strain and its reference Wistar-Kyoto strain. The DVL-1 sequence in the two strains was identical. Our results essentially exclude the DVL-1 gene as the cause for sensitivity to cerebral ischaemic insult in this rat model of stroke.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Electrophoretic analysis of neuronal genomic DNA from hypertrophic spinal ganglia during lizard tail regeneration.

Cytoplasmic and nuclear hypertrophy in neurons from the last 3 pairs of sensory ganglia left in situ cranially to the plane of amputation occurs during lizard tail regeneration. Cytophotometry after Feulgen staining demonstrated the presence of some neurons, from hypertrophic ganglia, whose quantity of DNA exceeded the diploid level (hyperdiploid neurons). In the present work agarose gel electrophoresis of total genomic DNA extracted from hypertrophic ganglia showed one or two bands migrating below the high molecular weight DNA, pointing to a selective amplification of discrete DNA segments.

Amputation, Surgical↗
PubMed2002

Overexpression and biochemical characterization of beta-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus influenzae strain Rd.

The lipopolysaccharide of capsule deficient Haemophilus influenzae strain Rd contains an N-acetylgalactosamine residue attached to the terminal globotriose moiety in the Hex5 glycoform. Genome analysis identified an open reading frame HI1578, referred to as lgtD, whose amino acid sequence shows significant level of similarity to a number of bacterial glycosyltransferases involved in lipopolysaccharide biosynthesis. To investigate its function, overexpression and biochemical characterization were performed. Most of the protein was obtained in a highly soluble and active form. By using standard glycosyltransferase assay and HPLC, we show that LgtD is an N-acetylgalactosaminyltransferase with high donor substrate specificity and globotriose is a highly preferred acceptor substrate for the enzyme. The K(m) for UDP-GalNAc and globotriose are 58 microM and 8.6 mM, respectively. The amino acid sequence of the enzyme shows the conserved features of family II glycosyltransferases. This is the first N-acetylgalactosaminyltransferase identified from H. influenzae, which shows potential application in large-scale synthesis of globo-series oligosaccharides.

Amino Acid Sequence↗
PubMed1997

Prevalence of Pro250Arg mutation of fibroblast growth factor receptor 3 in coronal craniosynostosis.

BACKGROUND: The C749G (Pro250Arg) mutation in the gene for fibroblast growth factor receptor 3 (FGFR3) has been found in patients with various types of craniosynostosis. We aimed to find out the proportion of cases of apparently non-syndromic coronal craniosynostosis attributable to this mutation. METHODS: We studied 26 patients with coronal craniosynostosis but no syndromic diagnosis, who were referred to a supra-regional specialist centre. Genomic DNA was analysed by PCR and restriction-enzyme digestion to identify the C749G mutation in FGFR3. Family members of patients found to have the mutation were also tested. FINDINGS: Eight (31%) of the 26 probands were heterozygous for the C749G mutation. In two cases, the mutation showed autosomal dominant transmission with evidence of variable expressivity; the remaining six cases were sporadic. We demonstrated in six families that the mutation had arisen de novo from clinically unaffected parents. INTERPRETATION: The C749G mutation in FGFR3 is a frequent cause of apparently non-syndromic coronal craniosynostosis. Our finding will aid genetic counselling and prenatal diagnosis. The mutation rate at this nucleotide is one of the highest described in the human genome.

Child, Preschool↗
PubMed1996

Phenotypic and genotypic characterization of clinically recovered presumptive Acinetobacter baumannii isolates which failed to grow at 44 degrees C.

Eleven clinical isolates of Acinetobacter, which exhibited an identical biochemical profile compatible with genospecies 3 and failed to grow at 44 degrees C, were not agglutinated by polyclonal rabbit immune sera against 26 serovars of genospecies 3. Rather, all 11 isolates reacted strongly with antiserum against serovar 18 of A. baumannii. Macrorestriction (SmaI) analysis of genomic DNA revealed that only one isolate was genotypically different, whereas the remaining ones were either closely related or identical. However, the genomic DNA of the A. baumannii serovar 18 reference strain proved to be genotypically unrelated.

Acinetobacter↗
PubMed2003

Quartz crystal microbalance (QCM) affinity biosensor for genetically modified organisms (GMOs) detection.

A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.

Affinity Labels↗
PubMed2006

Simple urinary sample preparation for proteomic analysis.

Since the completion of the human genome sequence, attention has now focused on establishing reference maps of body fluids such as plasma and urine for detecting diagnostic markers of disease. Although some progress has been made, challenges still remain in the development of an optimal sample preparation method for proteomic analysis of urine. We have developed a simple and efficient urine preparation method for two-dimensional (2-D) gel electrophoresis which involves precipitation of proteins with simultaneous desalting. Acetonitrile precipitation produced 2-D gel separations with the highest resolution and the greatest number of protein spots compared to precipitation by other organic solvents. The method was applied to observe changes in the urinary proteome over a 6 week period and to establish a reference map of a healthy subject. A total of 339 proteins from 159 genes was identified from healthy male urine by peptide mass fingerprinting. The profiles of the urinary proteome at three times in 1 day and on four different days were compared and were found to vary in number and spatial location of the proteins on the map. The method was also shown to be applicable to the higher concentrations of protein found in the urine of an ovarian cancer subject. We have developed a facile and robust method for preparing urine for 2-D gels that will encourage further use of urine.

Adult↗
PubMed2002

Molecular characterization of AtNAM: a member of the Arabidopsis NAC domain superfamily.

The petunia NAM and ArabidopsisATAF1 and CUC2 genes define the conserved NAC domain. In petunia, loss-of-function nam mutants result in embryos that fail to elaborate shoot apical meristems (SAM), and nam seedlings do not develop shoots and leaves. We have isolated a NAC domain gene, AtNAM, from an Arabidopsis developing seed cDNA library. Expression of AtNAM mRNA is restricted primarily to the region of the embryo including the SAM. The AtNAM gene contains three exons and is located on Chromosome 1. In vivo assays in yeast demonstrate that AtNAM encodes a transcription factor and that the NAC domain includes a specific DNA binding domain (DBD). The AtNAM DBD is contained within a 60 amino acid region which potentially folds into a helix-turn-helix motif that specifically binds to the CaMV 35S promoter. The putative transcriptional activation domain is located in the C-terminal region of the protein, a highly divergent region among NAC domain-containing genes. The Arabidopsis genome contains 90 predicted NAC domain genes; we refer to these collectively as the AtNAC superfamily. The first two exons of all members of this superfamily encode the NAC domain. Most AtNAC genes contain three exons with the last exon encoding an activation domain. A subfamily of AtNAC genes contains additional terminal exons coding for protein domains whose functions are unknown.

Amino Acid Sequence↗
PubMed1996

Genetic localization of a locus controlling the intensity of infection by Schistosoma mansoni on chromosome 5q31-q33.

Three hundred million individuals are at risk of infection by schistosomes and around 200,000 die each year of this disease. Severe clinical disease in schistosomiasis is often the consequence of heavy infection which, in several endemic areas, are determined largely by the susceptibility/resistance of individuals. Previously, we reported evidence, based on a segregation analysis in Brazilian pedigrees, that intensity of infection by Schistosoma mansoni was influenced by a major gene, indicating that host genetic factors are probably critical in controlling schistosome infection and disease development. To localize this gene, referred to as SM1, we performed a genome-wide study on 142 Brazilian subjects belonging to 11 informative families Our results show a linkage to only one region, on chromosome 5q31-q33, with maximum two-point lod scores of +4.74 and +4.52 for D5S636 and the colony stimulating factor-1 receptor marker (CSF1R), respectively. This was corroborated by multipoint analysis, indicating a close proximity to CSF1R as the most likely location of SM1. This region contains several candidate genes encoding immunological molecules that were shown to play important roles in human protection against schistosomes.

Brazil↗