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At least 1,009 records · Page 56Linked to original sources
PubMed1997

Efficient lysis of CD44v7/8-presenting target cells by genetically engineered cytotoxic T-lymphocytes--a model for immunogene therapy of cervical cancer.

Variant proteins of the CD44 surface glycoprotein family are expressed on many different human tumors and their lymph node metastases. An epitope encoded by sequences of variant exons CD44v7 and v8 and recognized by the monoclonal antibody VFF17 is frequently detected in cervical cancer, whereas the normal cervical epithelium lacks expression of this epitope. We have developed an immunotherapeutic approach for cervical cancer based on the expression of this CD44v7/8 epitope. The single chain antigen-binding fragment of VFF17 was fused to a signal transducing protein (zeta-chain) of the T-cell receptor complex (TCR) and was introduced into a retroviral gene transfer vector. Gene transfer was applied to the murine cytotoxic T-cell line cl96. All recombinant clones expressed the fusion protein on their cell surface. Functionality of the recombinant fusion protein was tested by subjection of several recombinant clones to in vitro cytotoxicity assays. CD44v7/8-expressing target cells were killed efficiently by reprogrammed cl96 in an MHC-independent fashion, whereas CD44v7/ 8-negative cells were not affected. These transfected T cell lines will now be tested in vivo using immune-deficient mice bearing CD44v7/8-expressing tumors.

Amino Acid Sequence↗
PubMed1998

Age-related differences in the preparatory processes of motor programming.

This article investigates the mechanisms underlying the age-related differences in information processing in the production of motor responses, especially the development of feedforward mechanisms. No age-related differences have emerged from developmental studies aiming at analyzing motor programming. Nevertheless, age effects have seldom been studied in function of motor preparation. The aim of the present experiment was (1) to study age differences in motor preparation, and (2) to validate the early maturation of movement parameters specification. Two conditions were used (1) no advanced information on the movement to be made was given to the subject (neutral prime), and (2) advanced information on which direction should be followed was provided to the subject, allowing him to prepare a response based on a prime (primed condition). Four age groups were studied 6, 8, 10 and 22 years. Our results showed mainly an early maturation of the programming processes. More specifically, (1) beyond 6 years of age, children are capable of using the information provided by the prime to prepare their movement in advance, (2) costs and benefits of pre-programming do not vary significantly with age, (3) deprogramming-reprogramming of effector and direction is quite similar across the four age groups.

Adult↗
PubMed1995

Disparate replication properties of integrated and extrachromosomal forms of bovine papilloma virus in ID13 cells.

Bovine papillomavirus (BPV) previously has been reported to exist in transformed rodent cell lines as both chromosomally integrated and extrachromosomal forms. In the BPV-transformed mouse cell line ID13, extrachromosomal BPV molecules replicate throughout S phase of the cell cycle in a random choice mode. We report here that these replication properties were altered for chromosomally integrated BPV DNA in five independent ID13 subclones. In all of the subclones, the integrated BPV sequences, which had no detectable deletions or mutations, existed in head-to-tail tandem arrays that replicated once per cell cycle, predominantly late in S phase. In contrast, extrachromosomal BPV molecules present in other subclones of the same cell line replicated in the random choice mode observed previously for non-integrated BPV. Our results indicate that the replication origin of integrated BPV either is inactivated as a consequence of chromosomal insertion, leading to the replication of BPV from origins in the flanking chromosomal DNA, or alternatively is reprogrammed to function in a once-per-cell cycle mode predominantly late in S phase.

Animals↗
PubMed1994

Discoordinate regulation of contractile protein gene expression in the senescent rat myocardium.

The myocardium is a highly adaptive tissue, as evidenced by phenotypic alterations throughout development and under conditions of altered hemodynamic load. With pressure overload, the myocardium displays adult-to-fetal transitions in expression of contractile and non-contractile proteins. Most intriguing is the fact that many of these transitions are also observed in the senescent heart. The purpose of this work was to establish if the thin filament regulatory proteins, troponin I and troponin T, exhibit reexpression of early developmental isoforms, suggestive of coordinate reprogramming of contractile protein isoform expression. As a functional index of reexpression of the early isoform of troponin I, slow skeletal troponin I, myofibrils were isolated from 12 and 24-month-old Fischer 344 rat ventricles and assayed for myofibrillar ATPase activity at pH 7.0 and 6.5. Both preparations displayed rightward shifts in Ca-ATPase relationships with no differences between groups. SDS-PAGE and Western blot analysis showed that whereas myosin heavy chain expression underwent a transition to predominance of the early development isoform, beta-myosin heavy chain, there was no reexpression of the fetal isoforms of either troponin I or troponin T in the rat heart at 24 months of age. Northern blot analysis using cDNA probes specific for cardiac or slow skeletal troponin I also confirmed the lack of slow skeletal reexpression in the 24-month ventricle. These results are significant in that they demonstrate a lack of coordinate expression of contractile protein isoforms under myocardial adaptation to the aging process.

Adenosine Triphosphatases↗
PubMed2000

Downregulation and nuclear relocation of MLP during the progression of right ventricular hypertrophy induced by chronic pressure overload.

The cardiac LIM domain protein MLP plays a crucial role in the architecture and mechanical function of cardiac myocytes. Mice lacking the MLP gene develop cardiac hypertrophy, dilated cardiopathy and heart failure. We investigated whether downregulation of MLP is induced by pressure overload and contributes to the physiopathology of cardiac hypertrophy and failure. We studied this mechanism in rat right ventricles submitted to pulmonary arterial hypertension, because it is known that this ventricle is very vulnerable to the deleterious effects of pressure overload. During the progression of cardiac hypertrophy to failure over a 31 days period there was a dramatic decrease by 50% of the MLP transcripts level. Consistently, immunohistochemistry detected very weak protein signals in the cytoplasms of cardiomyocytes at the failing stage, but myocytes nuclei were heavily labeled. The nuclear relocation was confirmed by the immunodetection of MLP on the nuclear and cytosolic fractions. This nuclear localization is the hallmark of a retro-differentiated phenotype, since it has been observed only in differentiating myoblasts. These changes were associated with ultrastructural disorganization of the myofibrils similar to that observed in MLP -/- mice. Therefore, MLP dowregulation occurring during gene reprogramming may critically contribute to mechanical failure of the myocardium.

Animals↗
PubMed2000

Involvement of p38 mitogen-activated protein kinase in the induction of tolerance to hemorrhagic and endotoxic shock.

BACKGROUND: Exposure to sublethal hemorrhage (SLH) makes rats tolerant to subsequent hemorrhagic or septic shock and is associated with altered NF-kappaB activity. The purpose of this study was to explore whether changes in p38 mitogen-activated protein (MAP) kinase activity also occur in the induction of tolerance by SLH. METHODS: Rats were made tolerant by SLH or sham operation. Twenty-four hours later rats were exposed to lipopolysaccharide (LPS) or had peritoneal macrophages (Mphi) isolated. CNI-1493, a p38 MAP kinase inhibitor, or saline was given prior to SLH. Lungs were harvested 1 h after SLH or LPS and total protein was extracted. Peritoneal Mphi were stimulated with LPS (10 microg/ml) and total protein was isolated 1 h later. Active, dually phosphorylated p38 MAP kinase was determined by Western blot. Tumor necrosis factor (TNF) was measured in Mphi supernatants by enzyme-linked immunosorbent assay (ELISA) 18 h after LPS. RESULTS: SLH activated p38 MAP kinase in the lung and this was inhibited by CNI-1493. Twenty-four hours later, lung p38 MAP kinase activity increased to the same degree in tolerant and sham rats following LPS, but much more prominently in the CNI-1493 treated rats. There was no p38 activity in peritoneal Mphi at baseline, and similar to lung p38, LPS led to increased p38 activity which was most significant in Mphi from rats that received CNI-1493 prior to SLH. TNF production by tolerant Mphi in response to LPS was significantly (P < 0.05, t test) decreased and p38 inhibition with CNI-1493 at the time of SLH reversed the inhibitory effects of tolerance on TNF production. CONCLUSIONS: TNF production by tolerant Mphi following a second insult (LPS) is attenuated despite preservation of normal p38 MAP kinase activity. However, activation of this intracellular second messenger is a necessary step in the "cellular reprogramming" that occurs during the induction of tolerance by SLH.

Adaptation, Physiological↗
PubMed1998

A structured, minimal parameter model of the central nitrogen metabolism in Saccharomyces cerevisiae: the prediction of the behavior of mutants.

In order to enable future pathway engineering of a complex system, such as the nitrogen metabolism in yeast, mathematical modelling tools have to be developed. The stoichiometric and biochemical characteristics of the glutamate and glutamine nodes (the Central Nitrogen Metabolism, CNM) are qualitatively known. Quantitative knowledge about the dynamics of the network lacks and needs to be developed for metabolic reprogramming. A model-based-experiment approach is proposed in which the development of a model initiates new experiments of which the results then improve the model. As a first step in this iterative system identification cycle, recent experimental data, both qualitative and quantitative, obtained from defined studies on the CNM of the yeast Saccharomyces cerevisiae have been translated into an initial mathematical model. The model approach is based on a combination of Flux Analysis and simple enzyme kinetics. The model is constructed using nonlinear Ordinary Differential Equations and regulation of the synthesis and activity of key enzymes of the CNM is included. The parameters of the model are estimated with a constrained Least Squares algorithm using the steady-state and dynamic pulse data of a glutamine limited continuous culture. The resulting model describes a continuous culture of a wild-type strain correctly and in general the trends of the dynamic behaviour after both glutamine and ammonia pulses to this culture are good. Inclusion of countercurrent reactions and compartmentation in the model is essential for the descriptive quality of the model under dynamic conditions. It is clear that more experimental work is needed. The model indicates that the GOGAT/Glutamine Synthetase (GS) pathway plays a more important physiological stabilizing role in yeast than is generally assumed. New, model-based, experiments have to investigate the function of GOGAT, especially under dynamic conditions. Also redox cofactors and ATP have to be measured. The resulting model is validated with data of similar experiments with a GS-mutant. The quality of the prediction of the behaviour of the mutant is comparable to the descriptive property, which is a very promising result, taking into account the limited dataset compared to the system complexity.

Ammonia↗
PubMed2000

Dynamics of gene expression in rat hepatocytes under stress.

The response of cells to physical or biochemical stress involves concerted changes in the expression of a large number of genes encoding various functions. We have used a quantitative kinetic RT-PCR technique to follow the dynamics of changes in transcription factor and acute-phase mRNA levels in cultured rat hepatocytes subjected to either elevated temperature (40 degrees C) or exposure to the inflammatory cytokine interleukin-6. The profiles of transcription factor gene expression displayed rapid and coordinate regulation, attainment of new steady-states, transitions in some instances from up-regulation to down-regulation (or vice versa), and, for elevated temperature, multiple spikes of up-regulation. Transcripts of acute-phase genes generally displayed relatively small changes during the first few hours followed by more significant changes over the course of tens of hours (elevated temperature) to days (IL-6 exposure). These observations are all consistent with the notion of genetic reprogramming due to a network of interacting transcription factor proteins and transcripts. We utilized a simple transcription/translation model incorporating autoregulation to describe the dynamics of transcription factor gene expression. This model successfully described key features of the transcription factor dynamics, most notably the multiple spikes observed after exposure to elevated temperature. The dynamics of gene expression are rich in information that, with considerably more study, may eventually be exploited to provide insights into the interplay of genetic networks in regulating a variety of cellular responses.

Acute-Phase Reaction↗
PubMedDate not supplied

Troglitazone induces expression of PPARgamma in liver.

Troglitazone is an insulin sensitizer which affects a number of target tissues. It is believed to exert these effects primarily by binding to and activating the y-isoform of peroxisome proliferator-activated receptor (PPARgamma), which in turn regulates the expression of specific genes. However, in a number of target organs, such as liver, the levels of PPARgamma are low and other isoforms predominate. In the present study, we examined whether troglitazone induces the expression of PPARgamma, thereby sensitizing cells for the action of this drug. Treatment of isolated rat hepatocytes with troglitazone induced both the mRNA and protein levels of PPARgamma in a dose-dependent fashion, with maximal levels of induction being three- to fourfold. This induction was also observed using the 15-deoxy-delta12,14-prostaglandin J2, a known natural ligand for PPARgamma, whereas ligands specific for PPARalpha were without effect. The induction of PPARgamma expression by troglitazone was also observed in livers from rats fed a diet containing troglitazone. Troglitazone had no effect on the expression of the alpha- or beta-isoforms of PPAR, the more predominant liver isoforms. These results indicate that troglitazone produces a reprogramming of PPAR isoform content in liver, which may in part underlie the mechanism whereby troglitazone sensitizes the liver to the action of insulin and/or ameliorates hyperglycemia.

Animals↗
PubMed2001

Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing.

The functional repertoire of the human genome is amplified by the differential assortment of exons. Spliceosome-mediated RNA trans-splicing can mobilize these packets of genetic information to reprogram mRNAs. In principle, this process could repair defective transcripts in loss-of-function genetic disorders in humans. We developed a tractable lacZ repair system to serve as a model for these genetic disorders. Targeted pre-trans-splicing RNA molecules efficiently and specifically repaired mutated lacZ transcripts and restored enzymatic activity in human cells. The development of this model confirms the potential for spliceosome-mediated RNA trans-splicing in genetic repairs and provides a powerful tool for rational design and in vitro evolution of pre-trans-splicing molecules.

Cell Fractionation↗
PubMed1999

Cloning the laboratory mouse.

A brief account is given of early attempts to clone mammals (mice) by transferring cells (nuclei) of preimplantation embryos into enucleated oocytes, zygotes or blastomeres of two-cell embryos. This is followed by a brief review of recent successes using adult somatic cells: mammary gland cells for sheep, muscle cells for cattle and cumulus cells for mice. We have developed a technique for cloning the laboratory mouse by transferring cumulus cell nuclei into enucleated oocytes. With this technique, we have produced a population of over 80 cloned animals, and have carried the process over four generations. Development and fertility of these appear normal. However, the yield is very low; only approximately 1% of injected oocytes are carried to term. The challenge is now to understand the reason for this high loss. Is it a problem of technique, genomic reprogramming, somatic mutation, imprinting or incompatible cell cycle phases?

Animals↗
PubMed1999

Therapeutic cloning: needs and prospects.

There has been widespread speculation regarding the possibility of reprogramming human somatic nuclei by transfer into oocytes. Stem cells derived from such reconstructed embryos could theoretically provide a source of autologous tissue for therapeutic transplantation. However the biological, technical and ethical hurdles which must be overcome before this can be realised are considerable. Here we review the background and prospects for such work.

Animals↗
PubMed1999

Nuclear transfer in farm animal species.

CLONE 'a group of two or more individuals with identical genetic makeup derived, by asexual reproduction, from a single common parent or ancestor' (The Chambers Dictionary 1993, Chambers Harrap). The term clone was originally applied to plants but has subsequently been used in a much broader context to include a person or thing closely similar to another, a copy or replica. In animals, true clones, as defined above, may be produced by embryo splitting or blastomere separation either artificially, or as occurs naturally in the production of identical twins. In these individuals all of the components making up the individual, including nuclear genetic material (the genome) and other maternally derived factors are derived from a single unique embryo which is the result of sexual reproduction. The term clone has been applied to animals produced by the technique of nuclear transfer. In this asexual process, nuclear genetic material is transferred from a donor cell (karyoplast) into a recipient cell (cytoplast) from which the genetic material has been removed. In farm animals the cytoplast of choice is the matured oocyte (or unfertilised egg) thus the animals developing from this technique are not true clones as each cytoplast is often derived from a different animal. The resultant animals may therefore be more aptly described as 'genomic copies'. In mammals, successful development of embryos reconstructed by nuclear transfer was originally restricted to using early embryos as nuclear donors, however, recent progress has demonstrated successful development using nuclei from embryonic, foetal and adult derived cell populations. Numerous factors affect the development of embryos reconstructed by nuclear transfer including; the cell cycle stage of the recipient cell, the cell cycle stage of the donor nucleus, the differentiated state of the donor nucleus, activation of the recipient cell, the culture method. In addition, there are variations in success between species, these may be related to differences in organisation of the cytoskeleton and/or the meiotic spindle in the recipient cell,differences in cell cycle control during early development, the onset of zygotic transcription or differences in the metabolic requirements of early embryos in vitro. The aim of this article is to describe and discuss some of these factors in relation to the successful development of nuclear transfer reconstructed embryos and in particular to the 'reprogramming' or 'remodeling' of the donor genetic material to attain successful development.

Animals↗
PubMed2000

Chemokines and viruses: the dearest enemies.

The relation between viruses and the chemokine system is characterized by a complex blend of enmity and attraction. Chemokines are key regulators of innate and adaptive immune responses against invading microorganisms, including viruses. They act not only as immune system "traffic officers," controlling leukocyte migration under both physiological and pathological conditions, but also as fine orchestrators that modulate the induction, amplification, and cytokine-secretion pattern of antiviral responses. However, viruses have succeeded in turning the chemokine system into an ally. During the course of a long parallel evolution, viruses have captured from their hosts the genetic information for encoding chemokines and chemokine receptors and have reprogrammed it for evading the control of the immune system. Moreover, selected viral agents, most notably primate immunodeficiency retroviruses, have adopted chemokine receptors as essential gateways for entry into their target cells. The endogenous secretion of chemokines is thus emerging as an important in vivo mechanism of viral control, which is potentially inducible by effective vaccines. The deepening knowledge of the interactions between viruses and chemokines may lead to novel therapeutic and preventive strategies for the control of viral and inflammatory diseases.

Animals↗
PubMed2005

Bacterial virulence strategies that utilize Rho GTPases.

The ability to modify central host cellular functions is a major advantage to many bacterial pathogens that use such strategies as part of their virulence mechanisms. Small GTPases, including Rho GTPases, make particularly attractive targets for pathogens because of their central roles in modulating cellular functions such as cytoskeletal control. Such modifications of these GTPases can include direct chemical modification of the GTPase or interfacing with some of the regulatory elements associated with GTPase control. Pathogens use these alterations in GTPase functions for a variety of functions, including killing the host cell, mediating bacterial uptake into the host cell (invasion), reprogramming actin to form a lesion in host cells underlying adherent bacteria, to mediate intracellular survival by affecting intracellular trafficking, or to provide polymerized actin mechanisms to propel microbes around inside host cells and into adjacent cells. Collectively, these examples represent many key microbial virulence mechanisms that have led to a much deeper understanding of both microbial pathogens and GTPase functions.

Animals↗
PubMed2006

Breaking ignorance: the case of the brain.

Immunological self-tolerance is maintained through diverse mechanisms, including deletion of autoreactive immune cells following confrontation with autoantigen in the thymus or in the periphery and active suppression by regulatory cells. A third way to prevent autoimmunity is by hiding self tissues behind a tissue barrier impermeable for circulating immune cells. The latter mechanism has been held responsible for self-tolerance within the nervous tissue. Indeed, the nervous tissues enjoy a conditionally privileged immune status: they are normally unreachable for self-reactive T and B cells, they lack lymphatic drainage, and they are deficient in local antigen-presenting cells. Yet the immune system is by no means fully ignorant of the nervous structures. An ever-growing number of brain specific autoantigens is expressed within the thymus, which ensures an early confrontation with the unfolding T cell repertoire, and there is evidence that B cells also contact CNS-like structures outside of the brain. Then pathological processes such as neurodegeneration commonly lift the brain's immune privilege, shifting the local milieus from immune-hostile to immune-friendly. Finally, brain-reactive T cells, which abound in the healthy immune repertoire, but remain innocuous throughout life, can be activated and gain access to their target tissues. On their way, they take an ordered migration through peripheral lymphoid tissues and blood circulation, and undergo a profound reprogramming of their gene expression profile, which renders them fit to enter the nervous system and to interact with local cellule elements.

Animals↗
PubMed2006

Familial hydatidiform molar pregnancy: the germline imprinting defect hypothesis?

Imprinting is the uniparental expression of a set of genes. Somatic cells carry two haploid sets of chromosomes, one maternal and one paternal, while germ cells contain only one of the two forms of chromosomes, male or female. This implies that during early embryogenesis the cells committed for developing the future germ cell lineage, the primordial germ cells, which are diploid, have to undergo a total chromosome reprogramming process. This process is delicately controlled during gametogenesis to ensure that males and females have only their respective form of gametes. The machinery involved in this process is yet poorly defined. Familial hydatidiform molar (HM) pregnancy is an abnormal form of pregnancy characterized by hydropic degeneration of placental villi and abnormal, or absence of, embryonic development. To date, the molecular defect causing this condition is unknown. However, in a few studied cases, the presence of paternal methylation patterns on the maternal chromosomes was observed. In this chapter, we summarize what is known about methylation aberrations in HMs and examine more closely the proposed hypothesis of a maternal germline imprinting defect.

DNA Methylation↗
PubMed2006

The role of histone modifications in epigenetic transitions during normal and perturbed development.

Epigenetic mechanisms control eukaryotic development beyond DNA-stored information. DNA methylation, histone modifications and variants, nucleosome remodeling and noncoding RNAs all contribute to the dynamic make-up of chromatin under distinct developmental options. In particular, the great diversity of covalent histone tail modifications has been proposed to be ideally suited for imparting epigenetic information. While most of the histone tail modifications represent transient marks at transcriptionally permissive chromatin, some modifications appear more robust at silent chromatin regions, where they index repressive epigenetic states with functions also outside transcriptional regulation. Under-representation of repressive histone marks could be indicative of epigenetic plasticity in stem, young and tumor cells, while committed and senescent (old) cells often display increased levels of these more stable modifications. Here, we discuss profiles of normal and aberrant histone lysine methylation patterns, as they occur during the transition of an embryonic to a differentiated cell or in controlled self-renewal vs pro-neoplastic or metastatic conditions. Elucidating these histone modification patterns promises to have important implications for novel advances in stem cell research, nuclear reprogramming and cancer, and may offer novel targets for the combat of tumor cells, potentially leading to new diagnostic and therapeutic avenues in human biology and disease.

Animals↗