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At least 1,027 records · Page 57Linked to original sources
PubMed1984

Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus.

A promoterless trp-lac fusion fragment was inserted near one end of the bacterial transposon Tn5 in the correct orientation to fuse lacZ gene expression to promoters outside Tn5. The resulting transposon, Tn5 lac, retains the kanamycin-resistance gene of Tn5 and transposes in Escherichia coli at 6% the frequency of Tn5 to many different sites in a bacteriophage lambda target. Expression of beta-galactosidase, the product of the lacZ gene, from Tn5 lac insertions in phage lambda depends both on insertion into a transcription unit in the correct orientation and on the regulation of the promoter of the transcription unit, verifying that by transposition Tn5 lac can fuse lacZ expression to outside promoters. An insertion of Tn5 lac in bacteriophage P1 was isolated and used to introduce Tn5 lac into Myxococcus xanthus, a bacterium that undergoes multicellular development. Stable kanamycin-resistant transductants are obtained that contain no P1 DNA sequences but have Tn5 lac inserted at different sites in the Myxococcus chromosome. Individual transductants express different levels of beta-galactosidase. A chromogenic substrate of beta-galactosidase, 5-bromo-4-chloro-3-indolyl beta-D-galactoside, is toxic in Myxococcus when cleaved in large amounts. In principle, Tn5 lac could be used to assay transcription in any bacterium in which Tn5 can transpose and beta-galactosidase can be measured.

Bacteriophage lambda↗
PubMed2004

Effect of metalloprotease inhibitors on corneal allograft survival.

PURPOSE: The expression of Fas ligand (FasL) in the cornea is essential for corneal allograft acceptance in mice. Because the expression of FasL on the surface of cells is sensitive to cleavage with matrix metalloproteases (MMPs), this study examined whether inhibitors of MMPs would lead to increased FasL expression and improved corneal allograft survival. METHODS: Corneal endothelia derived from mice and humans were treated with MMP inhibitors, and FasL expression was examined. BALB/c mice were engrafted with C57BL/6 or C57BL/6-gld corneas and treated with an ointment containing the MMP inhibitor, doxycycline. Corneal allograft survival was monitored for 50 days. RESULTS: Corneal endothelial cells from both mice and humans displayed increased surface expression of FasL after treatment with MMP inhibitors. The increase in surface expression was further evidenced by the ability of these cells to kill Fas-expressing target cells. Mice treated with doxycycline after corneal allograft transplantation showed significantly prolonged allograft survival and an increase in the overall acceptance rate. CONCLUSIONS: MMP inhibitor treatment of cornea-derived endothelial cells results in increased FasL expression and function. MMP inhibitor treatment prolongs corneal allograft survival and results in a modest increase in corneal allograft acceptance.

Animals↗
PubMed1995

A phase I trial of B7-transfected or parental lethally irradiated allogeneic melanoma cell lines to induce cell-mediated immunity against tumor-associated antigen presented by HLA-A2 or HLA-A1 in patients with stage IV melanoma. NCI protocol T93-0161. BRMP protocol 9401.

T cell lines generated by primary in vitro stimulation with B7-expressing HLA-A2+ melanoma cells lyse HLA-A2+ melanomas, but not non-melanomas that are HLA-A2+. Other data have demonstrated lack of response of these T cell lines against non-HLA-A2 melanomas. These concepts are verified by data from MALME MEL, which is killed, and MALME FIB, which is not. In no case was lysis directed at targets expressing potential allo-antigens (except for HLA-A2+ melanomas). A19 and Aw33 have not been excluded as possible allo-targets (but no data suggests they are). In total, it appears that much of the lytic activity observed in the two T cell lines is directed against HLA-A2-restricted, melanoma-specific antigens.

Adult↗
PubMed1999

Characterization and binding of intracellular antibody fragments to the hepatitis C virus core protein.

The monoclonal antibody C7-50 binds to the HCV core protein with high sensitivity and specificity. The coding sequences of the variable domains of the antibody were determined following cDNA cloning of the Fab and sFv fragments. Subsequently, intracellular expression and binding of these antibody fragments to the HCV core protein as a potential antiviral approach were studied. There was high specificity and sensitivity of binding of bacterially expressed, recombinant C7-50 Fab to HCV core as measured by EIA and immunoblot. For expression in mammalian cells, the C7-50 antibody was subcloned in the sFv format by the introduction of a (Gly(4)Ser)(3) linker spaced between light and heavy chains. Northern and Western blot analysis as well as confocal microscopy established the targeted expression of the C7-50 sFv antibody fragment in the endoplasmic reticulum of transfected cells. The colocalization and intracellular binding of the antibody fragment to HCV core protein was confirmed by immunoprecipitation and subsequent immunoblot analysis. This study demonstrates that gene delivery of cDNA coding sequences inducing intracellular expression of C7-50 antibody fragments leads to binding of the antibody fragment to the HCV core protein within the secretory compartment of transfected cells. Intracellular immunization represents a promising antiviral approach to interfere with the life cycle of HCV.

Amino Acid Sequence↗
PubMed2006

Antigen presentation in autoimmunity and CNS inflammation: how T lymphocytes recognize the brain.

The central nervous system (CNS) is traditionally viewed as an immune privileged site in which overzealous immune cells are prevented from doing irreparable damage. It was believed that immune responses occurring within the CNS could potentially do more damage than the initial pathogenic insult itself. However, virtually every aspect of CNS tissue damage, including degeneration, tumors, infection, and of course autoimmunity, involves a significant cellular inflammatory component. While the blood-brain barrier (BBB) inhibits diffusion of hydrophilic (immune) molecules across brain capillaries, activated lymphocytes readily pass the endothelial layer of postcapillary venules without difficulty. In classic neuro-immune diseases such as multiple sclerosis or acute disseminated encephalomyelitis, it is thought that neuroantigen-reactive lymphocytes, which have escaped immune tolerance, now invade the CNS and are responsible for tissue damage, demyelination, and axonal degeneration. The developed animal model for these disorders, experimental autoimmune encephalomyelitis (EAE), reflects many aspects of the human conditions. Studies in EAE proved that auto-reactive encephalitogenic T helper (Th) cells are responsible for the onset of the disease. Th cells recognize their cognate antigen (Ag) only when presented by professional Ag-presenting cells in the context of major histocompatibility complex class II molecules. The apparent target structures of EAE immunity are myelinating oligodendrocytes, which are not capable of presenting Ag to invading encephalitogenic T cells. A compulsory third party is thus required to mediate between the attacking T cells and the myelin-expressing target. This review will discuss the recent advances in this field of research and we will discuss the journey of an auto-reactive T cell from its site of activation into perivascular spaces and further into the target tissue.

Animals↗
PubMed1999

Reversible activation of c-Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion.

The protooncogene c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to achieve this, we have targeted expression of a switchable form of the c-Myc protein to the skin epidermis, a well characterized homeostatic tissue. We show that activation of c-MycER in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is sufficient to induce papillomatosis together with angiogenesis--changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycER.

Animals↗
PubMed2003

Loss of p53 in craf-induced transgenic lung adenoma leads to tumor acceleration and phenotypic switch.

One of the most frequent malignancies in humans is lung adenocarcinoma.To develop novel diagnostic and therapeutic approaches for the management of this disease, animal models are required. We have used transgenic mice with lung-targeted expression of the CRaf kinase to evaluate genes altered frequently in human lung adenocarcinoma for their effect on tumor progression. Here we report that loss of p53 dramatically accelerates tumor development and induces a phenotypic switch in the target cell from cuboid to a nonciliated columnar morphology. Coexpression of lung epithelial cell markers surfactant protein C and Clara cell antigen suggests that tumor cell dedifferentiation could be involved in this process. The effect of p53 is specific, because loss of one of its target genes, p21(CIP1/WAF1), did not have this effect on cell phenotype although tumor latency was also reduced significantly. Neither loss of p53 nor p21 stimulated acquisition of the metastasis program beyond the stage of bronchiolar extension. This mouse model for pulmonary adenoma and adenocarcinoma should be very helpful for a better understanding of pathogenesis and treatment of this most deadly human cancer.

Adenocarcinoma↗
PubMed1993

Alloreactive cytotoxic T lymphocytes generated in the presence of viral-derived peptides show exquisite peptide and MHC specificity.

The nature of alloreactivity to MHC molecules has been enigmatic, primarily because of the observation that allogeneic responses are considerably stronger than syngeneic responses. To better determine the specificity potential of allogeneic responses, we have generated alloreactive CTL specific for exogenous, viral-derived peptide ligands. This approach allowed us to critically evaluate both the peptide- and MHC-specificity of these alloreactive T cells. Exploiting the accessibility of the H-2Ld class I molecule for exogenous peptide ligands, alloreactive CTL were generated that are specific for either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis virus (LCMV) peptides bound by Ld alloantigens. Peptide specificity was initially observed in bulk cultures of alloreactive CTL only when tested on peptide-sensitized T2.Ld target cells that have defective presentation of endogenous peptides. Subsequent cloning of bulk alloreactive CTL lines generated to MCMV yielded CTL clones that had exquisitely specific MCMV peptide recognition requirement. All of the MCMV/Ld alloreactive CTL clones were also exquisitely MHC-specific in that none of the CTL clones lysed targets expressing MCMV/Lq complexes, even though Lq differs from Ld by only six amino acid residues and Lq also binds the MCMV peptide. This observation clearly demonstrates that alloreactive CTL are capable of the same degree of specificity for target cell recognition as are syngeneic CTL in MHC-restricted responses.

Amino Acid Sequence↗
PubMed2004

Stem cell expression of the AML1/ETO fusion protein induces a myeloproliferative disorder in mice.

The t(8;21)(q22;q22) translocation, present in 10-15% of acute myeloid leukemia (AML) cases, generates the AML1/ETO fusion protein. To study the role of AML1/ETO in the pathogenesis of AML, we used the Ly6A locus that encodes the well characterized hematopoietic stem cell marker, Sca1, to target expression of AML1/ETO to the hematopoietic stem cell compartment in mice. Whereas germ-line expression of AML1/ETO from the AML1 promoter results in embryonic lethality, heterozygous Sca1(+/AML1-ETO ires EGFP) (abbreviated Sca(+/AE)) mutant mice are born in Mendelian ratios with no apparent abnormalities in growth or fertility. Hematopoietic cells from Sca(+/AE) mice have markedly extended survival in vitro and increasing myeloid clonogenic progenitor output over time. Sca(+/AE) mice develop a spontaneous myeloproliferative disorder with a latency of 6 months and a penetrance of 82% at 14 months. These results reinforce the notion that the phenotype of murine transgenic models of human leukemia is critically dependent on the cellular compartment targeted by the transgene. This model should provide a useful platform to analyze the effect of AML1/ETO on hematopoiesis and its potential cooperation with other mutations in the pathogenesis of leukemia.

Animals↗
PubMed2001

Disruption of integrin function in the murine myocardium leads to perinatal lethality, fibrosis, and abnormal cardiac performance.

The molecular mechanisms that regulate the cardiac hypertrophic response and the progression from compensated hypertrophy to decompensated heart failure have not been thoroughly defined. Alteration in cardiac extracellular matrix is a distinguishing characteristic of these pathological processes. Integrins, cell surface receptors that mediate cellular adhesion to the extracellular matrix, are signaling molecules that possess mechanotransduction properties. Therefore, we hypothesized that integrins are likely candidates to play an important role in cardiac function. To test this hypothesis, transgenic mice were constructed in which normal integrin function was disrupted by expression of a chimeric molecule encoding the transmembrane and extracellular domains of the Tac subunit of the IL-2 receptor, fused to the cytoplasmic domain of beta(1A) integrin (Tacbeta(1A)). Using the alpha myosin heavy chain promoter to target expression of this chimera to the cardiac myocyte, transgenic mice were generated that had varied levels of transgene expression. Multiple transgenic founders that expressed the transgene at high levels, died perinatally and exhibited replacement fibrosis. Lines that survived showed 1) hypertrophic changes concordant with reduction in endogenous beta(1) integrin levels, or 2) reduced basal contractility and relaxation as well as alterations in components of integrin signaling pathways. These data support an important role for beta(1) integrin in normal cardiac function.

Animals↗
PubMed1998

Bioaccumulation of heavy metals with protein fusions of metallothionein to bacterial OMPs.

In view of potential biotechnological applications, eukaryotic metallothioneins (MTs) have been expressed in Escherichia coli as fusions to membrane or membrane-associated proteins such as LamB, the peptidoglycan-associated lipoprotein protein (PAL) or a hybrid Lpp/OmpA carrier sequence. The use of different anchors enables the MT moiety to be targeted into various cell compartments thus bringing the metal-binding ability of the resulting hybrids to specific sites of the cell structure. To this end, both full-size and partial sequences of the human or mouse MTs have been genetically fused to: i) the permissive site 153 of the LamB sequence, which loops out the MT to the external medium; ii) the N-terminus of a PAL variant devoid of its N-terminal cystein, which targets expression of the fusion into the periplasm; and iii) the C-terminus of Lpp-OmpA, for anchoring the MT to the outer membrane protein as an N-terminal fusion. Each type of fusion presented a distinct behavior in terms of expression, stability and ability to endow E. coli cells an enhanced accumulation of Cd2+, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions. The expression in vivo of metalloproteins bound to bacterial envelope structures opens a way to design biomass with specific metal-binding properties.

Animals↗
PubMed2003

Bilateral congenital cataracts result from a gain-of-function mutation in the gene for aquaporin-0 in mice.

Cataract Tohoku (Cat(Tohm)) is a dominant cataract mutation that leads to severe degeneration of lens fiber cells. Linkage analysis showed that the Cat(Tohm) mutation is located on mouse chromosome 10, close to the gene for aquaporin-0 (Aqp0), which encodes a membrane protein that is expressed specifically in lens fiber cells. Sequence analysis of Aqp0 revealed a 12-bp deletion without any change in the reading frame, which resulted in a deletion of four amino acids within the second transmembrane region of the AQP0 protein. Targeted expression of the mutated Aqp0 caused lens opacity in transgenic mice, the pathological severity of which depended on the expression level of the transgene. The mutated AQP0 protein was localized to the intracellular and perinuclear spaces rather than to the plasma membranes of the lens fiber cells. The cataract phenotype of Cat(Tohm) is caused by a gain-of-function mutation in the mutated AQP0 protein and not by a loss-of-function mutation.

Amino Acid Sequence↗
PubMed2006

A frameshift in the coding region of a novel tomato class I basic chitinase gene makes it a pseudogene with a functional wound-responsive promoter.

A putative class I basic chitinase gene, assigned as psiBCH, was cloned from a tomato breeding line NC 24E. The gene contains a coding region with two introns. The predicted psiBCH open reading frame (ORF) is 971 bp and exhibits 81-88% identity at the nucleotide level with known class I basic chitinase genes from the Solanaceae family. However, the presence of a stop codon caused by a frameshift in the ORF of psiBCH makes it unusual among the other class I plant basic chitinases. This stop codon might be involved in the lower accumulation of fully spliced psiBCH RNA caused by nonsense-mediated decay (NMD), which is an RNA surveillance system universally found in eukaryotes. Sequence analysis of the 1883-bp 5'-flanking region of the psiBCH gene revealed the presence of potential wound-response promoter elements. To study the transcriptional regulation of the psiBCH gene, its 5'-flanking region containing the putative promoter was fused to the gus reporter gene and introduced into the tobacco genome via Agrobacterium tumefaciens-mediated transformation. Transgenic plants were functionally assayed for beta-glucuronidase activity. The psiBCH promoter drives the reporter gene expression in response to wounding stimuli. psiBCH promoter-GUS analysis indicates that wound-response of the tobacco transgene was rapid and localized in the wounded area following mechanical wounding. Therefore, our results suggest that the psiBCH promoter can provide targeted expression of genes, such as protease inhibitors in response to pest attack.

Agrobacterium tumefaciens↗
PubMed2004

Regulation of muscle fiber type and running endurance by PPARdelta.

Endurance exercise training can promote an adaptive muscle fiber transformation and an increase of mitochondrial biogenesis by triggering scripted changes in gene expression. However, no transcription factor has yet been identified that can direct this process. We describe the engineering of a mouse capable of continuous running of up to twice the distance of a wild-type littermate. This was achieved by targeted expression of an activated form of peroxisome proliferator-activated receptor delta (PPARdelta) in skeletal muscle, which induces a switch to form increased numbers of type I muscle fibers. Treatment of wild-type mice with PPARdelta agonist elicits a similar type I fiber gene expression profile in muscle. Moreover, these genetically generated fibers confer resistance to obesity with improved metabolic profiles, even in the absence of exercise. These results demonstrate that complex physiologic properties such as fatigue, endurance, and running capacity can be molecularly analyzed and manipulated.

Animals↗
PubMed2006

Identification of evolutionarily conserved regulatory elements in the mouse Fgf8 locus.

The secreted signaling molecule fibroblast growth factor 8 (Fgf8) is an essential component of certain embryonic signaling centers including the mid-hindbrain (isthmic) organizer, the first branchial arch (BA1), and the apical ectodermal ridge (AER). In these signaling centers Fgf8 transcripts are expressed in a dynamic and transient fashion, but the mechanism by which this highly specific expression pattern is established remains largely unknown. We used DNA sequence comparisons coupled to transgenic approaches to obtain insight into the structure and function of regulatory elements in the Fgf8 locus. First, a bacterial artificial chromosome (BAC) containing the mouse Fgf8 gene partially rescues the embryonic lethality of Fgf8-deficient mice and controls Fgf8-specific gene expression of a coinjected lacZ reporter transgene. Second, sequence comparison of vertebrate Fgf8 loci revealed evolutionarily highly conserved noncoding sequences that were unexpectedly located mainly 3' of the Fgf8 coding region. Third, in transgenic mice some of these elements were sufficient to target expression to the AER, tail bud, and brain, including the isthmic organizer, indicating that they may represent Fgf8 cis-acting elements. Collectively, these data identify novel regulatory elements of the Fgf8 gene sufficient to drive expression to regions of known Fgf8 activity.

Animals↗
PubMed1998

Hypoxia-inducible factor 1: master regulator of O2 homeostasis.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates essential homeostatic responses to reduced O2 availability in mammals. Recent studies have provided insights into the O2-dependent regulation of HIF-1 expression, target genes regulated by HIF-1, and the effects of HIF-1 deficiency on cellular physiology and embryonic development.

Animals↗
PubMed2006

Cannabinoid WIN 55,212-2 regulates TRPV1 phosphorylation in sensory neurons.

Cannabinoids are known to have multiple sites of action in the nociceptive system, leading to reduced pain sensation. However, the peripheral mechanism(s) by which this phenomenon occurs remains an issue that has yet to be resolved. Because phosphorylation of TRPV1 (transient receptor potential subtype V1) plays a key role in the induction of thermal hyperalgesia in inflammatory pain models, we evaluated whether the cannabinoid agonist WIN 55,212-2 (WIN) regulates the phosphorylation state of TRPV1. Here, we show that treatment of primary rat trigeminal ganglion cultures with WIN led to dephosphorylation of TRPV1, specifically at threonine residues. Utilizing Chinese hamster ovary cell lines, we demonstrate that Thr(144) and Thr(370) were dephosphorylated, leading to desensitization of the TRPV1 receptor. This post-translational modification occurred through activation of the phosphatase calcineurin (protein phosphatase 2B) following WIN treatment. Furthermore, knockdown of TRPA1 (transient receptor potential subtype A1) expression in sensory neurons by specific small interfering RNA abolished the WIN effect on TRPV1 dephosphorylation, suggesting that WIN acts through TRPA1. We also confirm the importance of TRPA1 in WIN-induced dephosphorylation of TRPV1 in Chinese hamster ovary cells through targeted expression of one or both receptor channels. These results imply that the cannabinoid WIN modulates the sensitivity of sensory neurons to TRPV1 activation by altering receptor phosphorylation. In addition, our data could serve as a useful strategy in determining the potential use of certain cannabinoids as peripheral analgesics.

Animals↗
PubMed2001

Evaluation of the 5'-flanking regions of murine glutathione peroxidase five and cysteine-rich secretory protein-1 genes for directing transgene expression in mouse epididymis.

Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice.

Animals↗