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PubMed2005

Increased copy number at 3p14 in breast cancer.

INTRODUCTION: The present study was conducted to investigate if chromosome band 3p14 is of any pathogenic significance in the malignant process of breast cancer. Genetic studies have implicated a tumour suppressor gene on chromosome arm 3p and we have proposed LRIG1 at 3p14 as a candidate tumour suppressor. The LRIG1 gene encodes an integral membrane protein that counteracts signalling by receptor tyrosine kinases belonging to the ERBB family. LRIG1 mRNA and protein are expressed in many tissues, including breast tissue. METHODS: In the present report we analysed the LRIG1 gene by fluorescence in situ hybridisation (FISH), LRIG1 mRNA by quantitative RT-PCR, and LRIG1 protein by western blot analysis. Two tumour series were analysed; one series consisted of 19 tumour samples collected between 1987 and 1995 and the other series consisted of 9 tumour samples with corresponding non-neoplastic breast tissues collected consecutively. RESULTS: The LRIG1 gene showed increased copy number in 11 out of 28 tumours (39%) and only one tumour showed a deletion at this locus. Increased LRIG1 copy number was associated with increased levels of LRIG1 mRNA (two of three tumours) and protein (four of four tumours) in the tumours compared to matched non-neoplastic breast tissue, as assessed by RT-PCR and western blot analysis. CONCLUSION: The molecular function of LRIG1 as a negative regulator of ERBB receptors questions the biological significance of increased LRIG1 copy number in breast cancer. We propose that a common, but hitherto unrecognised, breast cancer linked gene is located within an amplicon containing the LRIG1 locus at 3p14.3.

Breast Neoplasms↗
PubMed1994

RNA transcripts of the beta-thalassaemia allele IVS-2-654 C-->T: a small amount of normally processed beta-globin mRNA is still produced from the mutant gene.

IVS-2-654 C-->T is a common Chinese beta-thalassaemia mutation. Previous studies report that this mutation resulted in the formation of an abnormally spliced mRNA and the absence of detectable normal beta-globin mRNA, hence the mutation was considered to cause beta o-thalassaemia. We recently used the method of PCR amplified cDNA copies of circulating erythroid cell mRNA to analyse the mutant gene transcripts and found that this IVS-2-654 mutation does not abolish normal RNA processing entirely, but that a significant amount (over 15%) of normally processed beta-globin mRNA is produced. Microglobin chain biosynthetic analysis using the HPLC method showed that beta-globin chain was also present in the blood of patients with IVS-2-654 C-->T mutation. Accordingly, this mutant allele leads to a beta (+)-thalassaemia. Further, the methodology described in this paper provides a new approach towards the detection of RNA transcripts of beta-thalassaemia alleles as well as the study of gene expression in beta-thalassaemia and other genetic diseases.

Alleles↗
PubMed2003

Analysis of colorectal tumor progression by microdissection and comparative genomic hybridization.

This investigation aimed to identify patterns of copy number change in colorectal tumor progression from adenoma to liver metastasis. Fifty-three microdissected sub-regions from 17 cases of colorectal cancer were assigned to one of six histopathologically defined categories: coexisting adenoma, tumor above the muscularis layer, tumor within the muscularis layer, tumor extending through the bowel wall to serosal fat, lymph node metastasis, and liver metastasis. Microdissected samples were treated by a microwave processing step and then used as templates for universal PCR amplification. PCR products were fluorophore labeled and subjected to comparative genomic hybridization. Copy number changes were found in all samples, and every chromosome arm (excluding acrocentric short arms) was affected. More losses than gains were detected, but there were no significant differences between the numbers of changes seen in each category. Each individual sample revealed unique changes, additional to those shared within each case. The most frequently observed gains were of X and 12q. The most common losses were of 8p, 16p, 9p, 15q, 18q, and 10q. Nominally significant associations were observed between metastatic tumor and loss of 12q24.1 or 10p13-14, non-metastatic tumor and loss of 8q24.1, tumor extending to serosal fat and loss of 6q24-25 or gain of 4q11-13, tumor extending to serosal fat and metastatic lesions and loss of 4q32-34 or 22q11-12, and adenoma and loss of 15q24. Loss of 4q32-34 remained highly significant after correction for multiple testing. Adenoma was the only category not to show loss of 17p. These data reveal a genetically heterogeneous picture of tumor progression, with a small number of changes associated with advanced disease.

Adenocarcinoma↗
PubMed1990

A herpes simplex virus type 1 latency-associated transcript mutant reactivates with normal kinetics from latent infection.

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) accumulate in neuronal nuclei of latently infected ganglia. Explant reactivation kinetics of LAT deletion mutants in the mouse eye model have suggested a role for the LATs in the reactivation process. This report describes the construction and characterization of an HSV-1 strain HFEM mutant, TB1, disrupted within both copies of the LAT gene. TB1 contains a 440-base-pair segment of bacteriophage lambda DNA in place of a 168-base-pair deletion within the transcribed portion of the LAT gene. The 2.0-kilobase LAT was not produced after infection of tissue culture cells with TB1, but a 0.7- to 0.8-kilobase RNA was expressed. TB1 did establish latent infection after corneal inoculation as efficiently as the parental virus, and its reactivation kinetics from explanted ganglia were similar to those of HFEM. During latent infection with TB1, HSV-1 transcripts were not detectable. Rescuant virus (TB1-R) contained intact LAT genes, synthesized full-length LAT transcripts during productive infection in tissue culture, and reactivated from ganglionic explants of latently infected mice with normal kinetics. Thus, any function these transcripts have in the reactivation process appears to include the region between the putative LAT promoter and the disruption in TB1--a region of approximately 1,600 nucleotides, 800 of which encode the LATs.

Animals↗
PubMed1987

Immunocytochemical distribution in rat brain of putative peptides derived from thyrotropin-releasing hormone prohormone.

Processing of the TRH prohormone (Pro-TRH), a protein of approximately 26,000 mol wt, could yield 5 copies of TRH, as well as extended forms of TRH and several other non-TRH peptides. To determine whether some of these peptides are formed and transported by axons in the rat brain, we used antiserum to synthetic peptides corresponding to portions of pro-TRH. These included the N-tyrosyl analogs [Tyr0]prepro-TRH-(25-50) (pYE27) and [Tyr1]prepro-TRH-(53-74) (pYT22) contained within the N-terminal flanking region of the prohormone, the N-tyrosyl analog [Tyr0]prepro-TRH-(165-186) (pYS23), expanding the fourth progenitor sequence of TRH in the midportion of the prohormone, and the synthetic peptide pAC12 corresponding to the first 12 amino acids of the C-terminal flanking region or prepro-TRH-(208-219). All antisera showed staining in neuronal perikarya and processes in all regions of the brain previously demonstrated to immunostain for TRH, including dense innervation of the external zone of the median eminence. In addition, these antisera immunostained regions of the brain not previously immunopositive for TRH. Not all regions reactive with antiserum to [Tyr0]prepro-TRH-(25-50) were also recognized by anti-pYT, -pYS, and -pAC. These studies confirm the presence of the deduced non-TRH sequences within the TRH precursor and their formation and transport in vivo in the central nervous system. The presence of immunoreactivity in regions of the brain that do not contain TRH and the variability of immunostaining of the different antisera in some of these regions suggest regional preferential processing of pro-TRH to other peptides that may be biologically active.

Animals↗
PubMed1991

Expression and processing of the rooster protamine mRNA.

In situ hybridization in immature and mature testis sections shows that the rooster protamine mRNA is transcribed in the post-meiotic stages of spermatogenesis. Two distinct populations of rooster protamine mRNA are expressed as determined by Northern blot analysis. Since there are two copies of the chicken protamine gene per haploid genome, the question was raised of whether the two mRNA populations correspond to the two different genes or was a result of differential mRNA processing. The fact that the two genes differ only in one nucleotide (one extra A in the polyadenylation signal in the second locus) and that random sequencing of several cDNA clones has revealed only one poly-A tail addition site favors the hypothesis that the differences are due to mRNA processing. This is supported by 3' S1 mapping which shows a single poly-A tail addition site, which in turn suggests that the heterogeneity is due to differences in the length of the poly-A tail. The latter is confirmed by RNAse H digestion of mRNA-Oligo-dT hybrids which shows that the two mRNA populations (470 +/- 20 nucleotide (nt) and 430 +/- 20 nt respectively) are converted to a single population of 345 +/- 15 nt in good accordance with the poly-A tail site determined by S1 mapping (347 nt). Thus one species has a poly-A tail of 145 nt appearing in round spermatids and the second, a shorter tail of 105 nt present at the later stages of elongated spermatids.

Amino Acid Sequence↗
PubMed2000

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

Amino Acid Motifs↗
PubMed1994

Two acquired immunodeficiency syndrome-associated Burkitt's lymphomas produce specific anti-i IgM cold agglutinins using somatically mutated VH4-21 segments.

We analyzed the reactivity and the structure of the VH and VL segments of two IgM monoclonal antibodies (MoAbs) produced by spontaneously in vitro outgrowing cell lines, HBL-2 and HBL-3, established from two acquired immunodeficiency syndrome (AIDS) patients with Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL). These B-cell clones were representative of the respective neoplastic parental clones, as determined by immunophenotypic and molecular genetic analysis. The IgM MoAbs were highly specific for the i determinant on red blood cells (cold agglutinins), but bound none of the other eight self and nine foreign antigens (Ags) tested, including those most commonly recognized by natural antibodies or autoantibodies. Structural analysis showed that the IgM MoAb VH segment sequences were 93.5% and 84.2% identical with that of the germline VH4-21 gene, which encodes the vast majority of cold agglutinins that are specific for the i/l carbohydrate Ag and are produced under chronic lymphoproliferative conditions. The HBL-2 MoAb VH4-21 gene segment was juxtaposed with 20P3 and JH6 genes and paired with a V lambda 1 segment, the sequence of which was 95.5% identical to that of the germline Humlv117 gene; the HBL-3 MoAb VH4-21 gene segment was juxtaposed with DXP'1 and JH5 genes and paired with a V lambda 1 segment, the sequence of which was 86.7% identical to that of the germline Humlv1L1 gene. The high degree of conservation of the VH4-21 gene in the human population, the nature of the nucleotide differences in the expressed VH4-21 segments, and the presence of nucleotide substitutions in the HBL-2 and HBL-3 IgM MoAb JH and/or J lambda segments suggested that the MoAb V segments underwent a process of somatic hypermutation. This was formally shown in the HBL-3 MoAb VH segment, by differentially targeted polymerase chain reaction amplification of the HBL-3 MoAb-producing cell genomic DNA. In addition, cloning and sequencing of the genomic DNA from fibroblasts of the same patient whose neoplastic B cells gave rise to the HBL-3 cell line yielded a germline copy of the VH4-21 gene. Thus, the expression of VH4-21 gene products may be involved in a self Ag-driven process of clonal B-cell expansion and selection associated with BL in these AIDS patients.

Agglutinins↗
PubMed2002

Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control.

Partially-deleted mitochondrial DNA (DeltamtDNA) accumulates during aging of postmitotic tissues. This accumulation has been linked to decreased metabolic activity, increased reactive oxygen species formation and the aging process. Taking advantage of cell lines with heteroplasmic mtDNA mutations, we showed that, after severe mtDNA depletion, organelles are quickly and predominantly repopulated with DeltamtDNA, whereas repopulation with the wild-type counterpart is slower. This behavior was not observed for full-length genomes with pathogenic point mutations. The faster repopulation of smaller molecules was supported by metabolic labeling of mtDNA with [3H]thymidine during relaxed copy number control conditions. We also showed that hybrid cells containing two defective mtDNA haplotypes tend to retain the smaller one as they adjust their normal mtDNA copy number. Taken together, our results indicate that, under relaxed copy number control, DeltamtDNAs repopulate mitochondria more efficiently than full-length genomes.

Aging↗
PubMed1998

Identification of dnaX as a high-copy suppressor of the conditional lethal and partition phenotypes of the parE10 allele.

Termination of DNA replication, complete topological unlinking of the parental template DNA strands, partition of the daughter chromosomes, and cell division follow in an ordered and interdependent sequence during normal bacterial growth. In Escherichia coli, topoisomerase IV (Topo IV), encoded by parE and parC, is responsible for decatenation of the two newly formed chromosomes. In an effort to uncover the pathway of information flow between the macromolecular processes that describe these events, we identified dnaX, encoding the tau and gamma subunits of the DNA polymerase III holoenzyme, as a high-copy suppressor of the temperature-sensitive phenotype of the parE10 allele. We show that suppression derives from overexpression of the gamma, but not the tau, subunit of the holoenzyme and that the partition defect of parE10 cells is nearly completely reverted at the nonpermissive temperature as well. These observations suggest a possible association between Topo IV and the replication machinery.

Alleles↗
PubMed2006

[Efficient expression and purification of human beta-defensin-2 in E.coli].

OBJECTIVE: To demonstrate the possibility of high-level expression of bioactive human beta-defensin-2 (hBD2) in E.coli, and to purify the recombinant hBD2. METHODS: DNA fragment containing mature hBD2 coding region (smhBD2-cDNA) was amplified by PCR, multiple copies of smhBD2-cDNA were linked using Bgl II and BamH I enzymes, pET32-nsmHBD2-cDNA with 1, 2, 4, or 8 copies of smhBD2-cDNA was constructed. The soluble and insoluble hBD2 proteins were separated and analyzed by SDS-PAGE analysis. The soluble protein underwent a separation process containing affinity chromatography, enterokinase digestion and ion exchange chromatography to get the recombinant hBD2 peptide. The bioactivity of recombinant hBD2 was examined by bacteria-inhibition tests in liquid culture. RESULT: The plasmids pET32-nsmHBD2-cDNA with 1, 2, 4 copies of smhBD2-cDNA were constructed and the expressed soluble protein accounted for 52 %, 48 %, and 31 % respectively. The plasmids with 8 copies expressed mainly insoluble protein with few in soluble form. The growth of E.coli K12D31 was dramatically suppressed with a inhibition rate of 90 %, when the final concentration of recombinant hBD2 reached between 0.4 to 0.5 mug/ml. CONCLUSION: Fusion expression of human beta-defensin-2 with multiple joined genes in E.coli could increase the expression of hBD2.

Anti-Infective Agents↗
PubMed1997

Reconstructing labroid evolution with single-copy nuclear DNA.

Fifteen per cent of all living fishes are united in a single suborder (Labroidei) and display a dazzling array of behavioural and ecological traits. The labroids are considered monophyletic and members share a pharyngeal jaw apparatus (PJA) modified for crushing and processing prey. Outside of the explicitly functional PJA, there is no corroborative evidence for a monophyletic Labroidei. Here, we report the first molecular phylogenetic analysis of the suborder. Contrary to morphology-based phylogenies, our single-copy nuclear DNA data do not support labroid families as a natural group. Our data indicate that pharyngognathy has evolved independently among labroid families and that characters of the PJA are not reliable markers of perciform evolution. This work 'crushes' conventional views of fish phylogeny and should engender novel concepts of piscine life history evolution.

Animals↗
PubMed1982

Architecture of Limulus polyphemus hemocyanin.

The architecture of the 48-meric hemocyanin of the horseshoe crab Limulus polyphemus has been determined from electron micrographs of whole (48-mer) molecules and half- (24-mer) molecules. The assembly of hexamers of kidney-shaped subunits can produce two dodecameric enantiomorphs, designated as right and left. The assembly of 24-mers can again result in two enantiomorphs. By taking into account the rocking effect described by Van Heel and Frank [Van Heel, M., & Frank, J. (1981) Ultramicroscopy 6, 187-194], we deduced that the 24-meric half-molecule is made up of two copies of the left dodecameric enantiomorph. In addition, the two constituent dodecamers of the half-molecule are shifted with respect to a symmetric head-to-tail arrangement, which makes it possible to distinguish two different faces of the 24-mer, termed flip and flop. A model of the whole molecule was built from two copies of the 24-meric half-molecule. This model presents the four distinct views observed in the electron microscope (pentagon, ring, cross, and bowtie). In addition, the model shows the pentagonal view to exist in two varieties: symmetric and asymmetric. An analysis of electron micrographs presenting the pentagonal view by image processing using the statistical technique of correspondence analysis confirmed the existence of two types of pentagonal view, representing projections of a molecule built from two copies of the left 24-meric enantiomorph. In addition, the best fit between the averaged molecule images and the possible models was observed with a flop-flop inter 24-mer contact. The final model is shown in a series of stereo views produced by computer graphical techniques.

Animals↗
PubMed2006

The quality of pharmacologic care for adults in the United States.

BACKGROUND: Despite rising annual expenditures for prescription drugs, little systematic information is available concerning the quality of pharmacologic care for adults in the United States. We evaluated how frequently appropriate pharmacologic care is ordered in a national sample of U.S. residents. METHODS: The RAND/UCLA Modified Delphi process was used to select quality-of-care indicators for adults across 30 chronic and acute conditions and preventive care. One hundred thirty-three pharmacologic quality-of-care indicators were identified. We interviewed a random sample of adults living in 12 metropolitan areas in the United States by telephone and received consent to obtain copies of their medical records for the most recent 2-year period. We abstracted patient medical records and evaluated 4 domains of the prescribing process that encompassed the entire pharmacologic care experience: appropriate medication prescribing (underuse), avoidance of inappropriate medications (overuse), medication monitoring, and medication education and documentation. A total of 3,457 participants were eligible for at least 1 quality indicator, and 10,739 eligible events were evaluated. We constructed aggregate scores and studied whether patient, insurance, and community factors impact quality. RESULTS: Participants received 61.9% of recommended pharmacologic care overall (95% confidence interval 60.3-63.5%). Performance was lowest in education and documentation (46.2%); medication monitoring (54.7%) and underuse of appropriate medications (62.6%) performance were higher. Performance was best for avoiding inappropriate medications (83.5%). Patient race and health services utilization were associated with modest quality differences, while insurance status was not. CONCLUSIONS: Significant deficits in the quality of pharmacologic care were seen for adults in the United States, with large shortfalls associated with underuse of appropriate medications. Strategies to measure and improve pharmacologic care quality ought to be considered, especially as we initiate a prescription drug benefit for seniors.

Adult↗
PubMed2005

To repress: a note on an ambiguity of meaning.

An analysand used the word repress in a dream in an unusual way. In the dream, a record had been re-pressed: it was not the original disc but a copy. This manifest meaning of the word led associatively to more latent meanings. A kind of dialectical process ensued whereby, whenever the concept of repression came up, several meanings had to be considered to set the record straight. The classical way of thinking about repression had been augmented a little by this novel meaning that the analysand had stumbled on in his dream. Psychoanalytic process was enriched by this ongoing scrutiny of repression in theory and practice.

Humans↗
PubMed1996

C-Fos is not essential for apoptosis.

The transcription factor AP-1, made up of dimers of Fos and Jun proto-oncogene products, is involved in distinct cellular processes, including cell proliferation, differentiation and apoptosis. In this study, we have used mice in which both copies of the c-fos gene were disrupted by targeted mutagenesis in order to analyze how the apoptotic response was affected in these mice. We prepared primary cultures from the lymphoid organs, spleen and thymus, obtained from both wild-type and c-fos -/- mice and analyzed the induction of apoptosis in these cultures in the absence and presence of etoposide, an inducer of apoptosis in distinct cell types. Primary cultures from both organs, spleen and thymus, isolated from wild-type mice underwent apoptosis after 3 and 6 h of culture, respectively. Addition of etoposide enhanced the apoptotic response and c-fos mRNA levels in both spleen and thymic cells. Nevertheless, we found that induction of apoptosis in primary cultures of cells obtained from spleen and thymus of c-Fos-deficient mice was practically identical to that observed in wild-type mice. These results demonstrate that c-Fos is not essential for apoptosis and that cells lacking c-Fos may undergo normal apoptosis.

Animals↗
PubMed1998

Single word production in nonfluent progressive aphasia.

We present an experiment investigation of the spoken single word production of two patients with nonfluent progressive aphasia. In Experiment 1, a task effect (reading > repetition > naming) suggested that phonological information available from task stimuli facilitated the patients' speech production; a length effect reflected the increased difficulty of phonological processing required for long words compared with that required for shorter words. Experiment 2 compared repetition, reading, copying, and writing to dictation tasks and demonstrated that a correspondence between input and output modality also facilitated performance. Experiment 3 showed that the patients' access to appropriate phonology in reading was positively related to the degree of correlation between orthographic and phonological forms. These results are discussed with reference to an account of pathologically weakened connections between nodes in an interactive spreading activation model of speech production of the type described by Dell (1986).

Aged↗