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PubMed2004

Involvement of the Arabidopsis thaliana AtPMS1 gene in somatic repeat instability.

Mismatch repair (MMR) genes participate in the maintenance of genome stability in all organisms. Based on its high degree of sequence conservation, it seems likely that the AtPMS1 gene of Arabidopsis thaliana is part of the MMR system in this model plant. To test this hypothesis, we aimed to disrupt AtPMS1 function by over-expressing mutated alleles expected to result in a dominant negative effect. To create one mutant allele we substituted two amino acids in the MutL-box, and for the other mutant allele we deleted 87 amino acids comprising the whole MutL-box. Contrary to published reports in some eukaryotes, transgenic plants expressing these alleles did not exhibit a decrease in fertility nor any other visible phenotype. To examine the impact of these mutations on microsatellite instability, the phenotype most often observed in organisms defective in MMR, reporter lines containing a uidA (GUS) gene inactivated by the insertion of a synthetic microsatellite (G7 or G16) were used. GUS gene function in these lines can be restored following the loss of one base or the gain of two bases in the repetitive tract. This results in the observation of blue sectors on a white background following histochemical staining. In a subset of the transformants, a significant increase (2- to 28-fold) in microsatellite instability was observed relative to wild-type. This report shows that MMR function can be disrupted via a dominant negative approach, and it is the first report to describe the phenotypic consequence of disrupting the function of a MutL homolog in plants.

Alleles↗

PubMed2006

Fluorescent protein expression driven by her4 regulatory elements reveals the spatiotemporal pattern of Notch signaling in the nervous system of zebrafish embryos.

Notch activation inhibits neuronal differentiation during development of the nervous system; however, the dynamic role of Notch signaling in individual cell lineages remains poorly understood. We have characterized 3.4 kb 5'-regulatory sequence of a Notch target gene, her4, and used it to drive fluorescent gene expression in transgenic lines where the spatiotemporal pattern of Notch activation can be examined in vivo. The 3.4 kb her4 promoter contains five predicted Su(H) binding sites of which two proximal sites were confirmed to be required for Notch-mediated transcriptional activation. Without Notch, Su(H) effectively represses transcription regulated by the promoter. Analyses of transgenic zebrafish showed that while the expression of proneural genes and Notch activation were both critical for endogenous her4 expression, reporter gene expression was primarily regulated by Notch activity. This study also showed that her4 may be differently regulated in sensory cranial ganglia, where Notch activity is not essential for her4 expression and where Su(H) may repress her4 expression. The establishment of a reporter line with Notch-Su(H)-dependent fluorescent gene expression provides a tool to explore the complex role of Notch signaling in the development of vertebrate nervous system.

Animals↗

PubMed1998

Expression of a functional anaphylatoxin C3a receptor by astrocytes.

Human astrocyte cell lines reportedly contain a specific receptor for the complement anaphylatoxin C3a based on ligand-binding studies, functional responses, and RNA analysis by RT-PCR. Uptake of 125I-C3a by astrocytes was specific and reversible. Scatchard analysis indicated the presence of two classes of binding sites. High-affinity binding sites were abundantly expressed (20,000-80,000 sites per cell) with an estimated K(D) of 1-2 nM. Low-affinity binding sites with a K(D) of 209 nM were largely expressed (n > or = 4 x 10(6) sites per cell) and probably did not reflect a receptor-mediated binding, but rather an ionic interaction between C3a and the membrane. Analysis of astrocyte mRNA by RT-PCR with three different sets of primers covering 60% of the C3a receptor (C3aR) mRNA sequence indicated that glial C3aR was identical to the leukocytic one. Western blot analysis using a specific anti-C3aR evidenced a C3aR with a molecular mass of 60,000 Da. C3a and a superagonist peptide, E7, induced a transient increase of intracellular [Ca2+] in primary culture of astrocytes. Treatment of the ligands by carboxypeptidase B to eliminate the C-terminus Arg considerably decreased the [Ca2+] response. Moreover, flow cytometry experiments demonstrated the expression of C3aR on normal rat astrocyte membrane. This report brings new insight for the role of the complement system in the brain inflammation response.

Anaphylatoxins↗

PubMed2005

Persistent infection of betanodavirus in a novel cell line derived from the brain tissue of barramundi Lates calcarifer.

In order to obtain an in vitro system for studying the mechanism of persistent infection of fish nodavirus, a novel cell line (BB) was established from the brain tissue of a barramundi, Lates calcarifer, which had survived viral nervous necrosis disease. The cell line has been subcultured > 100 times. The persistence of fish nodavirus designated as barramundi brain nervous necrosis virus (BBNNV) in the BB cells was demonstrated by: (1) the detection of the infectious virus in the culture supernatants, (2) the detection of NNV nucleic acids extracted from the BB cells, (3) the positive result of immunochemical staining using an NNV-specific monoclonal antibody and (4) their resistance to infection by another fish nodavirus grouper NNV (GNNV). No temperature-sensitive mutants were detected in the culture supernatant of the BB cells. Neither truncated genome (RNA1 or RNA2) nor smaller coat protein was found in the purified BBNNV particles, suggesting that defective interfering particles were unlikely to be important in the NNV-persistent infection in the BB cells. The result of the neutralization test indicated that the 5 antigenic determinants, recognized by GNNV-specific neutralizing antibodies, also existed on the coat protein of BBNNV. The BB cell line is the first cell line reported to be persistently infected with NNV, and would be a useful model for understanding the mechanisms of NNV-persistent infection in vitro and in vivo.

Animals↗

PubMed1995

Ion-channel activities regulate transmembrane signaling in thymocyte apoptosis and T-cell activation.

Several examples have shown that plasma membrane ion channels (e.g., Ca2+ and K+ channels) make an important contribution to lymphocyte activation or thymocyte apoptosis. Here we report on the importance of these ion channels in the sensitivity or resistance of lymphoid cells to extracellular ATP-induced apoptosis. Thymocytes of Balb/c mice responded to extracellular ATP (ATPex) sensitively, with an immediate increase in the intracellular calcium level and later with an increased membrane permeability to low MW markers. Mature (medullary) thymocytes showed a higher sensitivity than did cortical thymocytes. Three human lymphoma cell lines, including SUPT13, a cell line reported to be sensitive to TcR/CD3 activation-induced apoptosis, showed a high resistance to ATPex action. These observations suggest that maturation/differentiation state-dependent activity or disappearance of early ATP-receptor operated signaling systems (including ion channels) are critical for the cells in developing towards apoptosis. Using the patch-clamp technique we demonstrated that bretylium tosylate (a particular K(+)-channel blocker) known as inhibitor of T-lymphocyte proliferation also influences the single-channel properties of voltage-gated K+ channels through depressing whole-cell K+ currents. This finding is yet another example underlying the importance of K+ channel activity in T-lymphocyte proliferation.

Adenosine Triphosphate↗

PubMed2003

Dissecting the regulation of the Drosophila cell death activator reaper.

Programmed cell death or apoptosis is crucial to normal development and tissue homeostasis in multicellular organisms. In many cases malfunctioning of apoptotic pathways has disastrous consequences for the organism, like in the case of cancer, AIDS or neurodegenerative diseases, which all are associated with the inappropriate regulation of programmed cell death. In Drosophila, patterns of programmed cell death have been studied, and it is known that the induction of apoptosis requires the products of three closely linked genes, reaper (rpr), head involution defective (hid) and grim. Although it has been shown that rpr, hid and grim induce apoptosis through similar mechanisms, it is clear that they are not functionally equivalent, since their transcripts are differentially expressed in response to different signals. In this study, I dissected the temporal and spatial expression of the apoptosis promoting gene rpr by generating a series of reporter lines, which recapitulate various aspects of the endogenous rpr expression. Understanding the regulatory logic of reaper transcription will help to advance our knowledge of apoptosis regulation in Drosophila and in other organisms.

Animals↗

PubMed1997

Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination.

Rat fibroblast cell lines with targeted disruptions of both c-myc gene copies were constructed. Although c-myc null cells are viable, their growth is significantly impaired. The absence of detectable N-myc or L-myc expression indicates that Myc function is not absolutely essential for cell viability. The c-myc null phenotype is stable and can be reverted by introduction of a c-myc transgene. Exponentially growing c-myc null cells have the same cell size, rRNA, and total protein content as their c-myc +/+ parents, but the rates of RNA and protein accumulation as well as protein degradation are reduced. Both the G1 and G2 phases of the cell cycle are significantly lengthened, whereas the duration of S phase is unaffected. This is the first direct demonstration of a requirement for c-myc in G2. The G0-->S transition is synchronous, but S-phase entry is significantly delayed. The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.

Animals↗

PubMed2005

Z-138 cell line was derived from a patient with blastoid variant mantle cell lymphoma.

The Z-138 cell line, reported in the journal in 1998, was derived from a patient who developed a leukemia initially classified as chronic lymphocytic leukemia in 1987. Splenectomy for massive involvement was required in 1998 and the neoplasm subsequently transformed to an aggressive, mature B-cell leukemia 2 years later. At time of transformation, the neoplasm had a complex karyotype, including the t(11;14)(q13;q32). In light of the extensive updates in lymphoma classification that have occurred since that time, we reviewed the slides of the patient's neoplasm. The initial peripheral blood and bone marrow aspirate smears and the spleen were involved by numerous small lymphocytes with mature chromatin. The last bone marrow specimen was involved by slightly larger, irregular lymphocytes with immature chromatin and a high mitotic rate. Immunohistochemical analysis performed on the spleen and last bone marrow for this report showed that the neoplastic cells over-expressed cyclin D1. According to the criteria of the current World Health Organization lymphoma classification, this neoplasm is best classified as mantle cell lymphoma, with blastoid transformation present in the terminal phase of disease.

Aged↗

PubMed1997

Long terminal repeat U3 length polymorphism of human foamy virus.

Size determination of the long terminal repeat (LTR) of an early (1985) and a more recent (1993) passage of wild-type human foamy virus (HFV) revealed that the virus has undergone substantial deletions in the U3 region upon replication in tissue culture. Two LTR deletion variants (HSRV1 and 2) have been characterized in the past and used to construct molecular clones which are replication competent in cell culture. We now report the molecular cloning, sequencing, and biological characterization of an HFV genome with full-length LTR (pHFV2). Sequence analysis revealed that the deletions in HSRV1 and 2 are nonrandom and probably occurred by misalignment during reverse transcription. The comparative analysis of HFV2 and the variant with the largest U3 deletion, HSRV2, revealed a differential ability to replicate in human cell cultures. While HSRV2 replicated faster in diploid human fibroblasts, cells which have been used extensively for amplification of HFV in the past, replication of HFV2 was faster in a lymphoblastoid cell line. Reporter gene assays indicated that the cell-type specific ability of the LTRs to respond to the viral transcriptional transactivator may be a likely, reason for the different growth properties of both viruses and for the occurrence of the HFV U3 deletions. In foamy virus-infected chimpanzees only the full-length type of LTR was observed; however, the HSRV1 deletion variant was detected as the dominating virus in an accidentally HFV-infected human.

Animals↗

PubMed1991

Production of high-titer helper virus-free retroviral vectors by cocultivation of packaging cells with different host ranges.

The titer of retroviral vectors can be increased by cocultivation of retrovirus packaging cells that produce a vector with packaging cells having a different host range. Multiple rounds of infection occur in such cultures, producing an amplification of vector copy number and titer. Production of a vector with a very high titer of over 10(10) CFU per ml of conditioned medium has been reported, although replication-competent helper virus was also present. Since helper-free virus is a requirement for many applications of retroviral vectors, we repeated this procedure with a modified vector and achieved a 2- to 10-fold amplification of vector titer in the absence of helper virus, up to 2 x 10(7) CFU/ml. We have also repeated these experiments with the same vector and methods described previously or have assayed virus from the high-titer vector-producing cell line reported previously and observed maximum titers of 10(8) CFU/ml, invariably accompanied by helper virus. Thus, while amplification of vector titer in the absence of helper virus is possible, some unexplained difference in the assays for virus titer must account for our inability to obtain the exceptionally high vector titers that were reported previously.

Animals↗

PubMed1980

Establishment of a human hepatocellular carcinoma cell line releasing hepatitis B virus surface antigen.

A new hepatocellular carcinoma cell line, DELSH-5, derived from operative wedge biospy from a HBsAg sero- and tissue-positive patient, has been continuously propagated in vitro for nearly 22 months. The cells not only resemble hepatocytes on light and electron microscopic examination but also possess biosynthetic markers of the latter such as albumin and alpha-foetoprotein which were demonstrated in the supernatant medium as well as in the tumour cell cytoplasm. Karyology of cloned cells shows moderate aneuploidy, the major model chromosome number being 61. Though in the initial few passages HBsAg could not be detected, from the 13th passage onwards this viral component could be consistently demonstrated in small amounts in the concentrated supernatant medium by the macro- and micro-ELISA techniques. The immunohistochemical techniques as well as electron microscopy have failed to demonstrate any virus component inside the cell. The cell line reported here is the third of its kind which will act as a useful laboratory model to obtain pure HBsAg and to study the hepatitis-B-virus--liver-cell interaction with particular reference to the oncogenic potential of the virus.

Antigens, Neoplasm↗

PubMed1993

Autophagic degradation of N-linked glycoproteins is downregulated in differentiated human colon adenocarcinoma cells.

The aim of the present study was to elucidate the mechanism responsible for the high mannose glycoprotein instability in undifferentiated HT-29 cells (a human colon cancer cell line) reported previously. The results presented here are consistent with lysosomal degradation of these molecular species. In addition inhibitors of the autophagic-lysosomal degradative pathway (3-methyladenine, okadaic acid and asparagine) dramatically block the degradation of proteins and N-linked glycoproteins in undifferentiated HT-29 cells. The main conclusions of this work are: 1- the autophagic-lysosomal pathway is responsible for the high mannose glycoprotein degradation in undifferentiated HT-29 cells; 2- this degradative pathway exists in differentiated cells but is greatly reduced (3.5-4 fold); 3- the HT-29 cell line is a new model to investigate the molecular regulation of autophagy.

Adenine↗

PubMed1992

Retinoic acid receptor isoform RAR gamma 1: an antagonist of the transactivation of the RAR beta RARE in epithelial cell lines and normal human keratinocytes.

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR beta RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR beta RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR alpha 1 and RAR beta 2 isoforms, but not by RAR gamam 1. On the contrary, this latter isoform behaved towards the RAR beta RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR alpha 1 and RAR beta 2. RAR gamma 1 also behaved as an antagonist of the transactivation produced by cotransfected RXR alpha. The natural RAR beta gene promoter or RAR beta RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR gamma 1. It was, however, possible to activate to a certain extent RAR beta RARE-reporter constructs in NHK by co-transfecting RAR alpha 1, RAR beta 2 or RXR alpha. The antagonist behavior of RAR gamma 1 towards the RAR beta RARE may explain why in certain cell types such as keratinocytes, RAR beta is neither expressed nor induced by RA.

Animals↗

PubMed2005

Neural cell adhesion molecule-secreting transgenic mice display abnormalities in GABAergic interneurons and alterations in behavior.

The extracellular region of the transmembrane neural cell adhesion molecule (NCAM-EC) is shed as a soluble fragment at elevated levels in the schizophrenic brain. A novel transgenic mouse line was generated to identify consequences on cortical development and function of expressing soluble NCAM-EC from the neuron-specific enolase promoter in the developing and mature neocortex and hippocampus. NCAM-EC transgenic mice exhibited a striking reduction in synaptic puncta of GABAergic interneurons in the cingulate, frontal association cortex, and amygdala but not hippocampus, as shown by decreased immunolabeling of glutamic acid decarboxylase-65 (GAD65), GAD67, and GABA transporter 1. Interneuron cell density was unaltered in the transgenic mice. Affected subpopulations of interneurons included basket interneurons evident in NCAM-EC transgenic mice intercrossed with a reporter line expressing green fluorescent protein and by parvalbumin staining. In addition, there appeared to be a reduction in excitatory synapses, as revealed by synaptophysin staining and apical dendritic spine density of cortical pyramidal cells. Behavioral analyses demonstrated higher basal locomotor activity of NCAM-EC mice and enhanced responses to amphetamine and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate compared with wild-type controls. Transgenic mice were deficient in prepulse inhibition, which was restored by clozapine but not by haloperidol. Additionally, NCAM-EC mice were impaired in contextual and cued fear conditioning. These results suggested that elevated shedding of NCAM perturbs synaptic connectivity of GABAergic interneurons and produces abnormal behaviors that may be relevant to schizophrenia and other neuropsychiatric disorders.

Amphetamine↗

PubMed2006

Dynamics of heart differentiation, visualized utilizing heart enhancer elements of the Drosophila melanogaster bHLH transcription factor Hand.

Drosophila melanogaster has become one of the important model systems to investigate the development and differentiation of the heart. After 24h after egg deposition (h AED), a simple tube-like organ is formed, consisting of essentially only two cell types, the contractile cardioblasts and non-myogenic pericardial cells. In contrast to the detailed knowledge of heart formation during embryogenesis, only a few studies deal with later changes in heart morphology and/or function. This is mainly due to the difficulties to carry out whole mount stainings in later stages without complicated dissections or treatments of the cuticle and puparium. In this paper we describe the identification of a hand genomic region, which is fully sufficient to drive GFP expression in heart cells of embryos, larvae, and adults. This serves as an initial step to understand the position of hand in the early regulatory network in heart development. Furthermore, we demonstrate that our newly created GFP reporter line is extremely useful to study postembryonic heart differentiation. For the first time we document heart differentiation in living animals throughout all developmental stages of Drosophila melanogaster, including embryogenesis, all three larval stages, metamorphosis, and the adult life with respect to pericardial cells and cardiomyocytes.

Animals↗

PubMed2006

Systematic identification and classification of adverse events in human research.

Widely accepted standards and safeguards for research participants now include systematic surveillance and recording of adverse events. In the absence of a uniform regulation or structure for such reporting, each institution must now establish suitable yet efficient procedures to accomplish this task. We report herein our single center experience with a customized data collection, storage and review system specifically designed to identify and react appropriately to adverse events. Adverse events are classified by each investigator using three criteria in specific order: seriousness, expectedness and relatedness to the investigational intervention. Once classified, events are entered into an online database that includes collation, retrieval and search capabilities. Events meeting specified criteria are reviewed and adjudicated on a weekly basis by The University of Connecticut Research Adverse Events Committee, which makes advisory recommendations to the hospital's two Institutional Research Boards ranging from modification of informed consent to study suspension. Three hundred and seventy-one serious adverse events from > 900 studies were reviewed in the previous academic year. Our system, which combines timely on-line reporting with regular surveillance, provides a potential model that meets the need for comprehensive yet practical adverse events assessment and reporting.

Adverse Drug Reaction Reporting Systems↗

PubMed2006

In vitro phototoxicity of 5-aminolevulinic acid and its methyl ester and the influence of barrier properties on their release from a bioadhesive patch.

Topical administration of excess exogenous 5-aminolevulinic acid (ALA) leads to selective accumulation of the potent photosensitiser protoporphyrin IX (PpIX) in neoplastic cells, which can then be destroyed by irradiation with visible light. Due to its hydrophilicity, ALA penetrates deep lesions, such as nodular basal cell carcinomas (BCCs) poorly. As a result, more lipophilic esters of ALA have been employed to improve tissue penetration. In this study, the in vitro release of ALA and M-ALA from proprietary creams and novel patch-based systems across normal stratum corneum and a model membrane designed to mimic the abnormal stratum corneum overlying neoplastic skin lesions were investigated. Receiver compartment drug concentrations were compared with the concentrations of each drug producing high levels of PpIX production and subsequent light-induced kill in a model neoplastic cell line (LOX). LOX cells were found to be quite resistant to ALA- and M-ALA-induced phototoxicity. However, drug concentrations achieved in receiver compartments were comparable to those required to induce high levels of cell death upon irradiation in cell lines reported in the literature. Patches released significantly less drug across normal stratum corneum and significantly more across the model membrane. This is of major significance since the selectivity of PDT for neoplastic lesions will be further enhanced by the delivery system. ALA/M-ALA will only be delivered in significant amounts to the abnormal tissue. PpIX will only then accumulate in the neoplastic cells and the normal surrounding tissue will be unharmed upon irradiation.

Aminolevulinic Acid↗

PubMed1999

Cloning and characterization of the murine PKC alpha promoter: identification of a retinoic acid response element.

Protein kinase C (PKC) is a family which consists of multiple isoforms whose distinct physiological roles within the cell are unknown. We have previously demonstrated that levels of PKC alpha mRNA, protein, and enzyme activity in B16 melanoma cells can be modulated by retinoic acid. We investigated this regulation by cloning and characterizing the promoter region of the murine PKC alpha gene. A 13 kb mouse genomic fragment containing the 5' flanking region, first exon, and first intron was isolated and sequenced. Two transcription initiation sites were identified at 919 and 925 bp upstream from the translation start site. The promoter region contained a TATA-like box at -93 bp upstream of the transcription start site, but no CAAT box. Promoter activity differed between cell lines and correlated with the levels of PKC alpha expressed in these cell lines. Reporter gene assays showed that the region between -179 and -452 bp likely contains a silencer element(s). The promoter activity of a -179 bp fragment in B16 cells was stimulated twofold by retinoic acid. Within this region (-93 to -65 bp) there is a retinoic acid response element. An oligonucleotide spanning this region specifically bound exogenous RAR-RXR heterodimers and endogenous RAR from B16 nuclear extracts. These results suggest that retinoic acid increases PKC alpha gene expression in B16 cells, at least in part, through direct transcriptional stimulation of its promoter.

Animals↗