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Biomedical subjects

C Dean

Publications and source records attributed to C Dean.

At least 73 records · Page 4Linked to original sources
PubMed1998

The transposition frequency of Tag1 elements is increased in transgenic Arabidopsis lines.

Tag1 was identified as a highly active endogenous transposable element in transgenic Arabidopsis thaliana Landsberg erecta plants carrying the maize transposable element Activator (Ac). Here, we describe experiments designed to determine the basis for the high activity of Tag1. The frequency of transposition of Tag1 elements was compared in lines containing or lacking Ac transposase to assess the effect of Ac transposase on Tag1 activity. Three populations of nontransgenic plants, including nontransformed regenerants, were also analyzed. The high level of activity of Tag1 did not correlate with the presence or absence of Ac transposase but was significantly higher in transgenic lines. This result was maintained through at least six generations after transformation. These data suggest that Tag1 transposition is stimulated by processes that occur during the Agrobacterium transformation and that thereafter remain active. Two Tag1 elements are tightly linked in the Landsberg erecta genome and map to the lower arm of chromosome 1. Tag1 elements were found in only a few A. thaliana ecotypes but were present in four other Arabidopsis species.

Arabidopsis↗
PubMed1998

p21waf1/cip1mda-6 expression in non-small-cell lung cancer: relationship to survival.

In order to clarify critical events during bronchial carcinogenesis, and to evaluate a possible prognostic role for p21 immunohistochemical detection, we assessed the immunohistochemical expression of p21 protein in 60 surgically resected non-small-cell lung cancers (NSCLCs) that had been investigated previously for their p53 protein status. We found that p21 protein was expressed in both normal and neoplastic tissue. In normal tissue, p21 immunoreactivity was detectable in a low percentage of well-differentiated cells. We found immunostaining for p21 in 80% of the investigated neoplasms. In 73.3% of the neoplasms, p21 was considered to be overexpressed. No relationship was found between p21 overexpression and tumor stage or tumor-nodal-metastatic (TNM) status. The histologic grading was slightly correlated with the p21 status (P = -0.51), with no significant differences noted between squamous carcinomas and adenocarcinomas. Survival percentage curves for our lung-cancer patients, based on a comparison of different p21 expression levels and constructed through a Kaplan-Meier analysis, showed significant differences in mean (P < 0.001) and overall (P < 0.001) survival time between patients of different p21 status, suggesting a favorable prognostic value of p21 immunostaining for NSCLC patients.

Adenocarcinoma↗
PubMed1998

EGFR blockade by tyrosine kinase inhibitor or monoclonal antibody inhibits growth, directs terminal differentiation and induces apoptosis in the human squamous cell carcinoma HN5.

Human squamous cell carcinomas frequently overexpress the epidermal growth factor receptor (EGFR) and this is often associated with poor prognosis in patients with these cancers. The high level of expression of the EGFR provides an important target for therapy and we and others have shown that monoclonal antibodies (mAbs) which block the activation of the receptor by the EGF family of ligands inhibit the growth of EGFR overexpressing tumours in vitro and induce the regression of established tumours grown as xenografts in athymic mice. Inhibitors of the tyrosine kinase associated with the EGFR have also been shown to block receptor activation and prevent tumour cell proliferation. Using the EGFR-overexpressing head and neck carcinoma cell line HN5, we have compared the biological consequences of treatment with an inhibitor of EGFR tyrosine kinase (PD153035) with anti-EGFR monoclonal antibodies (mAbs) ICR63 or ICR80. We found that both the anti-EGFR mAbs and the TK inhibitor produce similar biological changes namely, they inhibit the EGF and TGFá-induced tyrosine phosphorylation of the receptor and the growth in culture of HN5 cells. At concentrations above 100 nM, the TK inhibitor prevented the growth in culture of HN5 cells completely with an IC50 of 40 nM. With the anti-EGFR mAbs, growth of HN5 cells was inhibited completely at concentrations above 4 nM with an IC50 of 1 nM. More importantly we found that, like the anti-EGFR mAbs, treatment with the TK inhibitor directs HN5 cells to undergo terminal differentiation as monitored by the expression of cytokeratin 10. In addition, our results indicate that the growth inhibitory effects of the anti-EGFR agents also lead to induction of apoptosis as determined by 7-amino actinomycin D staining (7-AAD). We conclude that EGFR blockade by anti-EGFR mAbs or TK inhibitor influences the growth in culture of EGFR overexpressing tumours by directing terminal differentiation and inducing apoptosis.

Antibodies, Monoclonal↗
PubMed1998

Collinearity between a 30-centimorgan segment of Arabidopsis thaliana chromosome 4 and duplicated regions within the Brassica napus genome.

Arabidopsis thaliana (the model dicotyledonous plant) is closely related to Brassica crop species. Genome collinearity, or conservation of marker order, between Brassica napus (oilseed rape) and A. thaliana was assessed over a 7.5-Mbp region of the long arm of A. thaliana chromosome 4, equivalent to 30 cM. Estimates of copy number indicated that sequences present in a single copy in the haploid genome of A. thaliana (n = 5) were present in 2-8 copies in the haploid genome of B. napus (n = 19), while sequences present in multiple copies in A. thaliana were present in over 10 copies in B. napus. Genetic mapping in B. napus of DNA markers derived from a segment of A. thaliana chromosome 4 revealed duplicated homologous segments in the B. napus genome. Physical mapping in A. thaliana of homologues of Brassica clones derived from these regions confirmed the identity of six duplicated segments with substantial homology to the 7.5-Mbp region of chromosome 4 in A. thaliana. These six duplicated Brassica regions (on average 22 cM in length) are collinear, except that two of the six copies contain the same large internal inversion. These results have encouraging implications for the feasibility of shuttling between the physical map of A. thaliana and genetic maps of Brassica species, for identifying candidate genes and for map based gene cloning in Brassica crops.

Arabidopsis↗
PubMed1997

AAC-11, a novel cDNA that inhibits apoptosis after growth factor withdrawal.

Many growth factors and cytokines act as cellular survival factors by preventing programmed cell death (apoptosis). However, the specific genes and corresponding proteins that mediate survival are poorly defined. To identify potential survival genes, a cDNA library was prepared from murine fibroblasts and screened by a functional expression cloning approach. A 1023-bp cDNA, AAC-11, was identified that encodes a protein of approximately 25 kDa. The AAC-11 gene shows strong species conservation and is ubiquitously expressed in embryonic and adult tissues with multiple transcripts, as well as in various human tumor cell lines. The predicted protein contains a leucine zipper domain but lacks a DNA-binding domain. BALB/c3T3 fibroblasts that were stably transfected with AAC-11 cDNA were viable in serum-free medium for up to 12 weeks. The protective action of AAC-11 was abolished by mutation of leucines to arginines within the leucine zipper domain. We also isolated a longer AAC-11 cDNA that codes for up to an additional 290 amino-terminal amino acids but did not protect against apoptosis. The cDNA for human AAC-11 was identified and exhibits strong homology with the murine species and retains the leucine zipper domain. Western immunoblots of BALB/c3T3 cells using rabbit anti-AAC-11 polyclonal serum revealed a major native 55-kDa AAC-11 protein and a minor 25-kDa protein corresponding to the long and short forms of AAC-11 cDNA, respectively. In summary, we report a cDNA whose expression supports cell viability after withdrawal of growth factors. The corresponding native protein may function as a novel inhibitor of apoptosis.

Alternative Splicing↗
PubMed1997

A unique domain of pRb2/p130 acts as an inhibitor of Cdk2 kinase activity.

The Cdk2 kinase has long been known to be involved in the progression of mammalian cells past the G1 phase restriction point and through DNA replication in the cell cycle. The Rb family of proteins, consisting of pRb, p107, and pRb2/p130, has also been shown to monitor progression of G1 phase, mostly through their interaction with E2F family members. p107 is able to inhibit Cdk2 kinase activity through this interaction via a p21-related domain present in the C terminus of the protein. We show here that pRb2/p130 also possesses this activity, but through a separate domain. Moreover, we correlate the increased expression of pRb2/p130 during various cellular processes with the decreased kinase activity of Cdk2. We hypothesize that pRb2/p130 may act not only to bind and modify E2F activity, but also to inhibit Cdk2 kinase activity in concert with p21 in a manner different from p107.

Animals↗
PubMed1997

Mapping of carotid baroreceptor subtype projections to the nucleus tractus solitarius using c-fos immunohistochemistry.

This study has combined physiological pressure stimulation of carotid baroreceptors via a vascularly isolated carotid sinus and anodal block of baroreceptor afferent fibers in the carotid sinus nerve to examine the medullary projections of type I vs. type II (large A- vs. small A- and C-fiber afferent) baroreceptors. The control distribution of cells in the nucleus tractus solitarius expressing c-fos in response to physiological activation of carotid baroreceptors in the isolated sinus was compared to that during anodal block of large A-fibers in the carotid sinus nerve. Carotid baroreceptor stimulation primarily activated neurons in the ipsilateral commissural and medial subnuclei of the caudal nucleus tractus solitarius and the dorsolateral, dorsomedial and medial subnuclei in the intermediate and rostral levels of the nucleus tractus solitarius. Elimination of large A-fiber carotid baroreceptor afferents, during similar carotid baroreceptor stimulation resulted in a decrease in the number of cells expressing c-fos in the dorsomedial subnucleus of the rostral nucleus tractus solitarius. These data indicate that projections of larger A-fiber (type I) carotid baroreceptors are localized primarily to the rostral dorsomedial subnucleus, while those of smaller A- and C-fiber baroreceptors are more widely distributed to the commissural, medial and dorsal subnuclei of the nucleus tractus solitarius.

Afferent Pathways↗
PubMed1997

FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains.

A strong promoter of the transition to flowering in Arabidopsis is encoded by FCA. FCA has been cloned and shown to encode a protein containing two RNA-binding domains and a WW protein interaction domain. This suggests that FCA functions in the posttranscriptional regulation of transcripts involved in the flowering process. The FCA transcript is alternatively spliced with only one form encoding the entire FCA protein. Plants carrying the FCA gene fused to the strong constitutive 35S promoter flowered earlier, and the ratio and abundance of the different FCA transcripts were altered. Thus, FCA appears to be a component of a posttranscriptional cascade involved in the control of flowering time.

Amino Acid Sequence↗
PubMed1997

Methodology for selection of human antibodies to membrane proteins from a phage-display library.

We describe a simple antigen capture technique for the selection of a specific human antibody to p185erbB-2, a transmembrane glycoprotein, from a library of human Fab genes expressed on the surface of bacteriophage. Magnetic beads coated with the rat antibody ICR55 have been used to capture erbB-2 antigen from Triton X-100 extracts of SKOV3 cells. The antigen-coated beads have then been used to select bacteriophage displaying human Fab with affinity for p185erbB-2. After 4 rounds of selection, 65 phage clones were isolated which bound specifically to p185erbB-2 in a capture assay. Nine of the clones which gave the strongest reaction in an ELISA were selected for further development and the Fab genes were subcloned into the expression vector pUC119his6mycXba and electroporated into E. coli TG1. Colonies were grown, induced and the supernatants tested for the presence of secreted human Fab. Supernatants from two of the 9 clones contained human Fab and one of these bound specifically to erbB-2 in a capture assay, stained the membranes of the erbB-2 overexpressing cell lines BT474 and SKBR3 and immunoprecipitated a protein of molecular weight 185 000 kDa from SKOV3 cells. We conclude that a membrane antigen captured by specific monoclonal antibody can be used successfully to select phage displaying human antibodies specific for the antigen.

Animals↗
PubMed1997

A novel zinc finger protein is encoded by the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death.

Arabidopsis Isd1 mutants are hyperresponsive to cell death initiators and fail to limit the extent of cell death. Superoxide is a necessary and sufficient signal for cell death propagation. Thus, LSD1 monitors a superoxide-dependent signal and negatively regulates a plant cell death pathway. We isolated LSD1 via its map position. The predicted LSD1 protein contains three zinc finger domains, defined by CxxCxRxxLMYxxGASxVxCxxC. These domains are present in three additional Arabidopsis genes, suggesting that LSD1 defines a zinc finger protein subclass. LSD1 is constitutively expressed, consistent with the mutant phenotype. Alternate splicing gives rise to a low abundance mRNA encoding an extra five amino-terminal amino acids. We propose that LSD1 regulates transcription, via either repression of a prodeath pathway or activation of an antideath pathway, in response to signals emanating from cells undergoing pathogen-induced hypersensitive cell death.

Alternative Splicing↗
PubMed1997

Medical abortion with oral methotrexate and vaginal misoprostol.

OBJECTIVE: To evaluate the safety and efficacy of oral methotrexate and vaginal misoprostol for medical abortion. METHODS: A prospective multicenter trial involved 300 women up to 49 days' gestation seeking elective abortion. Subjects received methotrexate 50 mg orally followed 5-6 days later by misoprostol 800 micrograms vaginally. The misoprostol dose was repeated if abortion did not occur. RESULTS: Complete abortion occurred in 273 of 299 women (91.3%; 95% confidence interval [CI] 87.5, 94.2%); one woman was lost to follow-up. Abortion occurred within 8 days of the methotrexate in 233 women (77.9%; 95% CI 72.8, 82.5%); the remaining 13.4% of women who aborted did so after a delay of 23.5 +/- 9.8 days (median 23 days, range 10-45). Vaginal bleeding lasted an average of 15 and 11 days in immediate and delayed-success abortions, respectively. Complete abortion rates decreased linearly with increasing body surface area. After methotrexate and misoprostol administration, nausea was reported in 37% and 33%, vomiting in 11% and 18%, diarrhea in 12% and 18%, and subjective fever or chills in 15% and 31% of subjects, respectively. CONCLUSION: Oral methotrexate followed by vaginal misoprostol is effective for abortion and represents an acceptable alternative to intramuscular methotrexate in regimens for medical abortion.

Abortifacient Agents, Nonsteroidal↗
PubMed1997

Multiple repetitive DNA sequences in the paracentromeric regions of Arabidopsis thaliana L.

Nine repetitive DNA sequences, present in the haploid Arabidopsis thaliana genome in 7-300 copies, were hybridized in situ to metaphase and interphase chromosomes. Every sequence was detected on all five chromosome pairs, but was not evenly dispersed over the genome. Clusters of signals were found in particular regions of the centromeric heterochromatin, and each sequence showed a characteristic distribution pattern. Some sequences hybridized more strongly on different chromosomes, reflecting chromosome-specific amplification or the presence of homologous sequences. No hybridization signals could be detected on euchromatic regions. In situ hybridization on extended chromatin fibres showed that the pAL1 repeats are interrupted by another repetitive DNA sequence. A cosmid subclone (74A) contained a (GA)38 microsatellite motif, and hybridization with a (GA) oligonucleotide revealed that most of the hybridization sites of 74A correspond to the distribution of this microsatellite motif. The results show that the paracentromeric heterochromatin of A. thaliana chromosomes is composed not only of the tandemly arranged 180-bp repeat family pAL1/pAtMr, but also of some other repetitive sequences, thus giving a better understanding of the organization of sequences at the centromeres of A. thaliana.

Arabidopsis↗
PubMed1997

Description of 31 YAC contigs spanning the majority of Arabidopsis thaliana chromosome 5.

In order to generate a physical map of Arabidopsis thaliana chromosome 5, 142 molecular markers mapping chromosome 5 have been used in colony hybridization experiments with four Arabidopsis, ecotype Columbia, yeast artificial chromosome (YAC) libraries. This resulted in 634 YAC clones being anchored on chromosome 5. Southern blot analysis confirmed their positioning and provided data, which along with knowledge of the sizes of all the YAC clones, enabled the clones to be arranged into 31 contigs. Genetic mapping of markers located within 29 of these contigs on the Landsberg erecta/Columbia recombinant inbred lines allowed positioning of the contigs along the chromosome. A high proportion of the YAC clones were found to contain chimaeric inserts. The availability of this YAC contig map will accelerate chromosome-walking experiments, provide substrates for large-scale genomic sequencing projects and facilitate the mapping of new probes to this chromosome.

Arabidopsis↗
PubMed1997

Inefficient and incorrect processing of the Ac transposase transcript in iae1 and wild-type Arabidopsis thaliana.

As part of the analysis of the Arabidopsis mutant iae1-1 (increased Ac excision), quantitative studies of the Ac transposase transcript were conducted. The primary transcript of Ac contains three small introns (introns 1-3; mean size 89 bp) and one larger intron (intron 4; 387 bp). We analysed the splicing of intron 3 and intron 4 in wild-type Arabidopsis and the iae1-1 mutant. Our results demonstrate that the splicing of Ac introns 3 and 4 is inefficient (splicing efficiencies 57 and 30% respectively) compared with that of an intron of an endogenous Arabidopsis gene (PHYB intron 1; splicing efficiency 90%). The poor splicing efficiency of Ac intron 4 was found to correlate with aberrant processing. Steady state levels of total Ac transcript were higher in the iae1-1 mutant than wild-type, but the same aberrant processing occurred. The inefficient processing of Ac in Arabidopsis prompted us to construct an Ac element lacking introns (Ac::cDNA) in an attempt to increase transposition frequencies. Autonomous activity of the Ac::cDNA element was undetectable in Arabidopsis, despite its ability to transpose at high frequency in response to a strong transposase source (35S::transposase) in trans, and the demonstrable autonomy of the same element in tobacco. A number of smaller transcripts were detected in Arabidopsis lines containing Ac::cDNA or Ac. Analysis of these smaller transcripts revealed a high frequency of premature polyadenylation in exon 2 and splicing of cryptic introns.

Arabidopsis↗
PubMed1997

Comparative analysis of non-random DNA repair following Ac transposon excision in maize and Arabidopsis.

Ac/Ds transposable elements often leave short DNA rearrangements, or 'footprints,' at the sites where they excise. Previous studies at the maize waxy (wx) gene suggest that the DNA repair that forms transposon footprints is not random. Each excision site consistently displays a different, predominant repair product suggesting flanking DNA may influence footprint formation. We have expanded these studies to show that predominant end-joining products also form in association with Ac/Ds excision in Arabidopsis and that chromosomal location of the Ac-containing construct does not appear to influence this repair. The predominant repair product is identical in both maize and Arabidopsis for Ac elements with the same adjacent DNA sequences. However, a broader range of minor footprint types is observed in Arabidopsis, including footprints that are rare in maize, suggesting potential differences in the host proteins involved in either transposition, repair or both. The data also suggest that the sequences influencing footprint formation are within 39 bp 5' and 18 bp 3' of the transposon. These studies demonstrate that transgenic Ac/Ds-containing plants will be useful tools in dissecting plant DNA repair processes.

Arabidopsis↗
PubMed1997

Cardiovascular disease control efforts among local health departments in Missouri: four-year trends and policy implications.

Knowledge, attitudes, and activities in cardiovascular disease (CVD) control among local health departments in Missouri were surveyed in 1990 and in 1994, following four years of a community-based CVD risk-reduction project. Hypertension screening was rated as the most frequently performed CVD-related activity in 1990 and in 1994. Differences in mean scores between baseline and follow-up surveys showed no significant changes except for cholesterol screening. Respondents preferred individual patient education rather than community-wide approaches to risk reduction. Our findings highlight the need for more emphasis on coalition building and community-based programs for preventing and controlling CVD at the local health department level.

Cardiovascular Diseases↗
PubMed1997

The Arabidopsis downy mildew resistance gene RPP5 shares similarity to the toll and interleukin-1 receptors with N and L6.

Plant disease resistance genes operate at the earliest steps of pathogen perception. The Arabidopsis RPP5 gene specifying resistance to the downy mildew pathogen Peronospora parasitica was positionally cloned. It encodes a protein that possesses a putative nucleotide binding site and leucine-rich repeats, and its product exhibits striking structural similarity to the plant resistance gene products N and L6. Like N and L6, the RPP5 N-terminal domain resembles the cytoplasmic domains of the Drosophila Toll and mammalian interleukin-1 transmembrane receptors. In contrast to N and L6, which produce predicted truncated products by alternative splicing, RPP5 appears to express only a single transcript corresponding to the full-length protein. However, a truncated form structurally similar to those of N and L6 is encoded by one or more other members of the RPP5 gene family that are tightly clustered on chromosome 4. The organization of repeated units within the leucine-rich repeats encoded by the wild-type RPP5 gene and an RPP5 mutant allele provides molecular evidence for the heightened capacity of this domain to evolve novel configurations and potentially new disease resistance specificities.

Alleles↗