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At least 811 records · Page 45Linked to original sources
PubMed2000

Regulation of gut and heart left-right asymmetry by context-dependent interactions between xenopus lefty and BMP4 signaling.

The Lefty subfamily of TGFbeta signaling molecules has been implicated in early development in mouse, zebrafish, and chick. Here, we show that Xenopus lefty (Xlefty) is expressed both bilaterally in symmetric midline domains and unilaterally in left lateral plate mesoderm and anterior dorsal endoderm. To examine the roles of Xlefty in left-right development, we created a system for scoring gut asymmetry and examined the effects of unilateral Xlefty misexpression on gut development, heart development, and Xnr-1 and XPitx2 expression. In contrast to the unilateral effects of Vg1, Activin, Nodal, or BMPs, targeted expression of Xlefty in either the left or the right side of Xenopus embryos randomized the direction of heart looping, gut coiling, and left-right positioning of the gut and downregulated the asymmetric expression of Xnr-1 and XPitx2. It is currently thought that Lefty proteins act as feedback inhibitors of Nodal signaling. However, this would not explain the effects of right-sided Xlefty misexpression. Here, we show that Xlefty interacts with the signaling pathways of other members of the TGFbeta family during left-right development. Results from coexpression of Xlefty and Vg1 indicate that Xlefty can nullify the effects of Vg1 ectopic expression and that Xlefty is downstream of left-sided Vg1 signaling. Results from coexpression of Xlefty and XBMP4 indicate that XLefty and XBMP4 interact both synergistically and antagonistically in a context-dependent manner. We propose a model in which interactions of Xlefty with multiple members of the TGFbeta family enhance the differences between the right-sided BMP/ALK2/Smad pathway and the left-sided Vg1/anti-BMP/Nodal pathway, leading to left-right morphogenesis of the gut and heart.

Amino Acid Sequence↗
PubMed2000

Expression and membrane assembly of a transmembrane region from Neu.

Transmembrane domains of receptor tyrosine kinases are increasingly seen as key modulatory elements in signaling pathways. The present work addresses problems surrounding expression, isolation, secondary structure recovery, and assembly into membranes, of the relatively large quantities of transmembrane peptides needed to investigate these pathways by NMR spectroscopy. We demonstrate significant correspondence between SDS-PAGE behavior of such peptides and their (2)H NMR spectra in lipid bilayer membranes. A 50-residue peptide, Neu(exp), containing the transmembrane portion of the receptor tyrosine kinase, Neu, was designed for expression in Escherichia coli. The sequence also contained 11-12 amino acids from each side of the transmembrane domain. The common problem of low expressivity of transmembrane peptides was encountered-likely associated with membrane toxicity of the desired gene product. This difficulty was overcome by expressing the peptide as a TrpE fusion protein in a pATH vector to target expression products to inclusion bodies, and subsequently removing the TrpE portion by cyanogen bromide cleavage. Inclusion bodies offered the additional benefits of reduced proteolytic degradation and simplified purification. The presence of a hexa-His tag allowed excellent recovery of the final peptide, while permitting use of denaturing solvents and avoiding the need for HPLC with its attendant adsorption losses. Isolated expressed peptides were found to be pure, but existed as high oligomers rich in beta-structure as evidenced by CD spectroscopy and SDS-PAGE behavior. Dissolution in certain acidic organic solvents led to material with increased alpha-helix content, which behaved in detergent as mixtures of predominantly monomers and dimers-a situation often considered to exist in cell membranes. For purposes of NMR spectroscopy, peptide alanine residues were deuterated in high yield during expression. The same acidic organic solvents used to dissolve and dissociate expressed transmembrane peptides proved invaluable for their assembly into lipid bilayers. Analogous transmembrane peptides from the human receptor tyrosine kinase, ErbB-2, demonstrated related phenomena.

Amino Acid Sequence↗
PubMed2003

Differences in properties between human alphaA- and alphaB-crystallin proteins expressed in Escherichia coli cells in response to cold and extreme pH.

It has been reported that alphaA-crystallin has greater protective effects against apoptosis in lens epithelial cells than alphaB-crystallin [Andley, Song, Wawrousek, Fleming and Bassnett (2000) J. Biol. Chem. 275, 36823-36831]. Because the alphaA-crystallin proteins are specifically expressed in the vertebrate lens, we examine the non-specific properties of both alphaA- and alphaB-crystallins in an Escherichia coli system. E. coli cells were transformed with the inducible protein expression vector pET-11a, harbouring the gene for either human alphaA- or alphaB-crystallin, and two other control plasmids, pET-1la vector alone or pGEX-2T vector encoding GST (glutathione S-transferase). These cells were exposed to various stress conditions, such as cold-shock at 4 degrees C or extremely low or high pH environments (pH 4.7 or pH 8.0) for 6 h, and survival of the host cells and the solubility of the expressed target proteins in the cytosol were examined. Under these stress conditions, the cells expressing alphaB-crystallin protein demonstrated significantly improved survival when compared with the other cells, and the expressed protein in the cytosol was almost soluble, in contrast with the alphaA-crystallin protein. Differences in the amino acid sequence between the proteins in a phenylalanine-rich region next to the N-terminal consensus alpha-crystallin domain was considered to be responsible for chaperone activity and cell survival.

Amino Acid Sequence↗
PubMed2004

Imaging endoplasmic reticulum calcium with a fluorescent biosensor in transgenic mice.

The use of biosynthetic fluorescent sensors is an important new approach for imaging Ca(2+) in cells. Genetically encoded indicators based on green fluorescent protein, calmodulin, and fluorescence resonance energy transfer (FRET) have been utilized to measure Ca(2+) in nonmammalian transgenic organisms and provide information about the organization and regulation of Ca(2+) signaling events in vivo. However, expression of biosynthetic FRET-based Ca(2+) indicators in transgenic mammals has proven to be problematic. Here, we report transgenic expression of an endoplasmic reticulum (ER) Ca(2+) biosensor in mouse pancreas. We targeted expression of a yellow cameleon3.3er (YC3.3er) transgene with mouse insulin I promoter. YC3.3er protein expression was limited to pancreatic beta-cells within islets of Langerhans and absent in the exocrine pancreas and other tissues. Animals developed and matured normally; sensor expression was unaffected by age. Glucose tolerance in transgenic mice was also unaffected, indicating the transgenic biosensor did not impair endocrine pancreas function. ER Ca(2+) responses after administration of thapsigargin, carbachol, and glucose were measured in individual beta-cells of intact islets using confocal microscopy and confirmed the function of the biosensor. We conclude that controlling transgene transcription with a cell-specific promoter permits transgenic expression of FRET-based Ca(2+) sensors in mammals and that this approach will facilitate real-time optical imaging of signal transduction events in living tissues.

Animals↗
PubMed2003

Suprabasal alpha6beta4 integrin expression in epidermis results in enhanced tumourigenesis and disruption of TGFbeta signalling.

Inappropriate alpha6beta4 integrin expression correlates with a high risk of tumour progression in stratified squamous epithelia. Targeted expression of alpha6beta4 in the suprabasal layers of transgenic mouse epidermis dramatically increased the frequency of papillomas, carcinomas and metastases induced by chemical carcinogenesis, independent of the beta4 cytoplasmic domain. Suprabasal alpha6beta4 also perturbed transforming growth factor beta (TGFbeta) signalling as demonstrated by decreased nuclear Smad2 in transgenic epidermis and tumours. In cultured keratinocytes, suprabasal alpha6beta4 relieved TGFbeta-mediated growth inhibition and blocked nuclear translocation of activated Smad2/3. Responsiveness to TGFbeta could be restored by inhibiting cadherin-mediated cell-cell adhesion or phosphoinositide 3-kinase (PI3-K) activity, but not by inhibiting mitogen-activated protein kinase (MAPK) activity. These data suggest that suprabasal alpha6beta4 promotes tumourigenesis by preventing TGFbeta from suppressing clonal expansion of initiated cells in the epidermal basal layer.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2001

Soluble guanylate cyclase is required during development for visual system function in Drosophila.

A requirement for nitric oxide (NO) in visual system development has been demonstrated in many model systems, but the role of potential downstream effector molecules has not been established. Developing Drosophila photoreceptors express an NO-sensitive soluble guanylate cyclase (sGC), whereas the optic lobe targets express NO synthase. Both of these molecules are expressed after photoreceptor outgrowth to the optic lobe, when retinal growth cones are actively selecting their postsynaptic partners. We have previously shown that inhibition of the NO-cGMP pathway in vitro leads to overgrowth of retinal axons. Here we examined flies mutant for the alpha subunit gene of the Drosophila sGC (Gcalpha1). This mutation severely reduced but did not abolish GCalpha1 protein levels and NO-stimulated sGC activity in the developing photoreceptors. Although few mutant individuals possessed a disorganized retinal projection pattern, pharmacological NOS inhibition during metamorphosis increased this disorganization in mutants to a greater degree than in the wild type. Adult mutants lacked phototactic behavior, and the off-transient component of electroretinograms was frequently absent or greatly reduced in amplitude. Normal phototaxis and off-transient amplitude were restored by heat shock-mediated Gcalpha1 expression applied during metamorphosis but not in the adult. We propose that diminished sGC activity in the visual system during development causes inappropriate or inadequate formation of first-order retinal synapses, leading to defects in visual system function and visually mediated behavior.

Animals↗
PubMed2006

A novel role for a Drosophila homologue of cGMP-specific phosphodiesterase in the active transport of cGMP.

cGMP was first discovered in urine, demonstrating that kidney cells extrude this cyclic nucleotide. Drosophila Malpighian tubules provide a model renal system in which a homologue of mammalian PDE (phosphodiesterase) 6 is expressed. In humans, this cG-PDE (cGMP-specific PDE) is specifically expressed in the retinal system, where it controls visual signal transduction. In order to gain insight into the functional role of DmPDE6 (Drosophila PDE6-like enzyme) in epithelial function, we generated transgenic animals with targeted expression of DmPDE6 to tubule Type I (principal) cells. This revealed localization of DmPDE6 primarily at the apical membranes. As expected, overexpression of DmPDE6 resulted in elevated cG-PDE activity and decreased tubule cGMP content. However, such targeted overexpression of DmPDE6 creates a novel phenotype that manifests itself in inhibition of the active transport and efflux of cGMP by tubules. This effect is specific to DmPDE6 action, as no effect on cGMP transport is observed in tubules from a bovine PDE5 transgenic line which display reduced rates of fluid secretion, an effect not seen in DmPDE6 transgenic animals. Specific ablation of DmPDE6 in tubule principal cells, via expression of a targeted DmPDE6 RNAi (RNA interference) transgene, conferred increased active transport of cGMP, confirming a direct role for DmPDE6 in regulating cGMP transport in tubule principal cells. Pharmacological inhibition of DmPDE6 in wild-type tubules using the cG-PDE inhibitor, zaprinast, similarly results in stimulated cGMP transport. We provide the first demonstration of a novel role for a cG-PDE in modulating cGMP transport and efflux.

Animals↗
PubMed1996

Mucin-1-related T cell infiltration in colorectal carcinoma.

Mucins (MUC) are highly glycosylated molecules widely expressed on epithelia of different origins, including colonic mucosa. Altered glycosylation processes in tumour cells result in the exposure of normally cryptic peptide epitopes, which may then be recognized as tumour-specific antigens. Recently, MUC1-specific antibodies were detected in the serum of a broad range of cancer patients, and from different tumours tumour-specific cytotoxic T lymphocytes (CTL) were isolated that recognized MUC1. Absence of HLA restriction in the recognition has been ascribed to the highly repetitive sequence of the polypeptide core, allowing simultaneous recognition of multiple identical epitopes and cross-linking and aggregation of T cell receptor on mucin-specific T cells. We investigated the expression of MUC1 epitopes in 56 cell suspensions from Dukes' B to D colorectal carcinomas using antibodies that recognize distinct peptide sequences on the glycosylated or deglycosylated MUC1 protein backbone. No relation was observed between MUC1 expression, or the extent of its glycosylation, and Dukes' stage, tumour location and tumour differentiation, but a positive correlation was detected between the percentages of tumour cells expressing mucin-1 and the numbers of CD3+ infiltrating cells. These tumour-infiltrating lymphocytes contained, however, only a few MUC1-specific T lymphocytes, as CTL showing preferential killing of MUC1-expressing target cells were only obtained from one tumour. Since, in addition, the majority of colorectal carcinomas were found to express the fully glycosylated MUC1 glycoprotein, its potential role as a target antigen for T-lymphocyte-mediated immunotherapy in this tumour type is probably limited.

Antibodies, Monoclonal↗
PubMed2004

Expression of Vitreoscilla haemoglobin in tobacco cell cultures relieves nitrosative stress in vivo and protects from NO in vitro.

Targeted expression of Vitreoscilla haemoglobin (VHb) has been analysed in Nicotiana tabacum plants and suspension cultures under various growth and stress conditions. VHb localization to different cell compartments (cytoplasm, chloroplast and mitochondria) was successful, as judged by signal peptide cleavage. The presence of VHb in subcellular compartments did not result in phenotypical differences between these plant lines. In contrast with previous reports, we were unable to discern any significant changes in growth and other phenotypical characteristics between VHb-expressing and transformed control plants under standard growth conditions. When exposed to nitrosative stress, growth of VHb-expressing cultures was less affected relative to transformed controls. Furthermore, a diminished inactivation of the NO-sensitive enzyme aconitase was observed in the presence of VHb. In contrast, no protective effect of VHb expression against oxidative stress could be detected.

Journal Article↗
PubMed1994

DNA regulatory sequences of the rat tyrosine hydroxylase gene direct correct catecholaminergic cell-type specificity of a human growth hormone reporter in the CNS of transgenic mice causing a dwarf phenotype.

Transgenic mice bearing 4.8 kilobases (kb) of upstream rat tyrosine hydroxylase (TH) sequences linked to a human growth hormone gene (hGH) exhibited cell-specific expression of hGH in all the appropriate catecholaminergic neurons in the central nervous system (CNS), although with different penetrance in two different mouse lineages. No ectopic expression was observed in any brain or peripheral region in one founder and its progeny. In another founder there was some ectopic expression in addition to appropriate and high levels of tissue-specific expression in all catecholaminergic areas. These results identify regulatory sequences that are sufficient for targeting expression to all catecholaminergic CNS neurons. Also, expression of exogenous hGH in the hypothalamus caused a dwarf phenotype, generating a novel genetic model for GH deficiency of hypothalamic origin.

Aging↗
PubMed2005

Puralpha activates PDGF-A gene transcription via interactions with a G-rich, single-stranded region of the promoter.

Transcription of the PDGF-A chain gene is regulated by multiple promoter and silencer elements that are GC-rich and exhibit considerable single-stranded character. In this study, the 42 kDa single-stranded DNA and RNA binding protein, Puralpha, was investigated with respect to its ability to bind and interact functionally with single-stranded DNA elements in the PDGF-A gene. Recombinant GST-Puralpha bound with high affinity and sequence-specificity to the G-rich strands of two such transcriptional control elements, the 5'-S1 nuclease-hypersensitive silencer (5'SHS; -1418 to -1388) and the nuclease-hypersensitive element (NHE; -92 to -48). Ethylation interference footprinting localized binding of Puralpha to a region between nucleotides -91 and -77 within the NHE element, which contains binding sites for the double-stranded DNA-binding transcription factors Sp1, EGR-1 and WT1. Forced expression of Puralpha upregulated transcriptional activity of the PDGF-A promoter but not the 5'SHS silencer in HepG2 cells, demonstrating Puralpha has the potential to activate PDGF-A gene expression. Targeted disruption of the Puralpha gene reduced NHE activity and PDGF-A mRNA expression in mouse embryo fibroblasts, consistent with a physiological role for Puralpha in maintaining optimal transcription of the PDGF-A gene. These results indicate Puralpha enhances transcription of the PDGF-A gene through its interactions with single-stranded, G-rich strands in the promoter, perhaps by stabilizing non-B-form DNA conformations.

Animals↗
PubMed2006

Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor.

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

Animals↗
PubMed2004

Sonic hedgehog and insulin-like growth factor signaling synergize to induce medulloblastoma formation from nestin-expressing neural progenitors in mice.

Medulloblastoma (MB) is a malignant brain tumor that arises in the cerebellum of children. Activation of the Sonic hedgehog/Patched (Shh/Ptc) signaling pathway in neural progenitor cells of the cerebellum induces MBs in mice. The incomplete penetrance of tumor formation in mice, coupled with the low frequency of mutations in Shh/Ptc pathway genes in human tumors, suggests that other signaling molecules cooperate with Shh to enhance MB formation. We modeled the ability of insulin-like growth factor (IGF) signaling to induce MB using the RCAS/tv-a system, which allows postnatal gene transfer and expression in a cell-type-specific manner. We used RCAS retroviral vectors to target expression of Shh, IGF2, and activated Akt to nestin-expressing neural progenitors in the cerebella of newborn mice. The incidence of Shh-induced tumor formation (15%) was enhanced by coexpression with IGF2 (39%) and Akt (48%). Neither IGF2 nor Akt caused tumors when expressed independently. The induced tumors showed upregulated expression of insulin receptor substrate 1 and phosphorylated forms of IGF1 receptor and Akt, mimicking activated IGF signaling found in human MBs. These results indicate that combined activation of the Shh/Ptc and IGF signaling pathways is an important mechanism in MB pathogenesis.

Animals↗
PubMed2004

Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis.

To develop murine models of leukemogenesis, a series of transgenic mice expressing BCR-ABL in different hematopoietic cell subsets was generated. Here we describe targeted expression of P210 BCR-ABL in stem and progenitor cells of murine bone marrow using the tet-off system. The transactivator protein tTA was placed under the control of the murine stem cell leukemia (SCL) gene 3' enhancer. Induction of BCR-ABL resulted in neutrophilia and leukocytosis, and the mice became moribund within 29 to 122 days. Autopsy of sick mice demonstrated splenomegaly, myeloid bone marrow hyperplasia, and extramedullary myeloid cell infiltration of multiple organs. BCR-ABL mRNA and protein were detectable in the affected organs. Fluorescence-activated cell sorter (FACS) analysis demonstrated a significant increase in mature and immature myeloid cells in bone marrow and spleen, together with increased bilineal B220+/Mac-1+ cells in the bone marrow. tTA mRNA was expressed in FACS-sorted hematopoietic stem cells expanded 26-fold after BCR-ABL induction. Thirty-one percent of the animals demonstrated a biphasic phenotype, consisting of neutrophilia and subsequent B-cell lymphoblastic disease, reminiscent of blast crisis. In summary, this mouse model recapitulates many characteristics of human chronic myeloid leukemia (CML) and may help elucidate basic leukemogenic mechanisms in CML stem cells during disease initiation and progression.

Animals↗
PubMed1994

GFAP promoter directs astrocyte-specific expression in transgenic mice.

Glial fibrillary acidic protein (GFAP) is an intermediate-filament protein expressed abundantly and almost exclusively in astrocytes of the CNS. We are studying transcriptional regulation of the GFAP gene to gain insight into astrocyte function and also to develop an astrocyte-specific expression system for manipulating brain physiology. In this work, we have produced transgenic mice carrying the bacterial lacZ reporter gene linked to a 2.2 kilobase 5'-flanking sequence derived from the human GFAP gene that previously was shown to direct astrocyte-specific transcription in cultured cells. We report that this promoter directs expression to astrocytes in the CNS. In addition, the upregulation of GFAP gene activity that follows injury to the brain was mimicked by the transgene. One of the transgenes was found to be X-linked and appeared to undergo the usual random inactivation that achieves gene dosage compensation in females. The brains of hemizygous females stained uniformly rather than displaying mosaic patches, indicating that astrocytes intermingle following their formation. The specific expression of the GFAP-lacZ transgene means that it is now possible to target expression of other heterologous genes to astrocytes in vivo, and to study the mechanisms for reactive gliosis at the DNA level.

Animals↗
PubMed2002

Tumor-targeted gene therapy for nasopharyngeal carcinoma.

The unique feature of human nasopharyngeal carcinoma (NPC) is its almost universal association with the EBV, which is expressed in a latent form exclusively in cancer cells, and not in the surrounding tissues. We have exploited this differential by constructing a novel replication-deficient adenovirus vector (ad5.oriP) in which transgene expression is under the transcriptional regulation of the family of repeats domain of the origin of replication (oriP) of EBV. When EBNA1, one of the latent gene products of EBV, binds to the family of repeats sequence, this activates transcription of downstream genes. Vector constructs were made using the beta-galactosidase and luciferase reporter genes (ad5oriP.betagal and ad5oriP.luc) or the p53 tumor suppressor gene (ad5oriP.p53). 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining demonstrated extensive expression only in EBV-positive NPC cells, specifically in response to the presence of EBNA1. The relative difference in expression between EBV-positive and -negative cell lines is approximately 1000-fold. This selective expression was corroborated in EBV-positive and -negative tumor models, along with an absence of transgene expression in the host liver. Significant cytotoxicity was achieved using the adv.oriP.p53 therapeutic gene only in EBV-positive NPC cells, which was enhanced with the addition of ionizing radiation. Cytotoxicity was mediated primarily by induction of apoptosis. These results demonstrate that the oriP sequence can achieve high levels of gene expression targeted specifically to EBV-positive NPC cells in the context of the adv vector. This has now provided the tumor-specific expression system from which additional interventions can be evaluated in future treatment strategies for patients with nasopharyngeal cancers.

Adenoviridae↗
PubMed2001

DNA tumor vaccines.

A new generation of vaccines are being developed to induce immune responses that fight off infectious agents, or erradicate cancerous cells. These new vaccines are based on a plasmid vector, which in transfected mammalian cells cause constitutive high-level expression of the target antigen. Expression of the target antigen, in turn, can induce a full-range of immunologic responses, including cell-mediated killing, cell-mediated cytokine release and the production of antigen-specific antibodies. Through molecular techniques, these nucleic acid vaccines can be enhanced to increase target antigen expression and facilitate antigen presentation. Additionally, genetic adjuvants expressed simultaneously with the target antigens can induce the immune responses to disease-associated antigens. The ease with which these genetic vaccines can be generated and the potency of their ability to generate immune-mediated responses make them highly effective, which creates hope for developing effective treatment and prevention of various diseases, most notably cancer.

Adjuvants, Immunologic↗
PubMed2001

Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced apoptosis in colorectal cancer cells.

Loss of p53 gene function, which occurs in most colon cancer cells, has been shown to abolish the apoptotic response to 5-fluorouracil (5-FU). To identify genes downstream of p53 that might mediate these effects, we assessed global patterns of gene expression following 5-FU treatment of isogenic cells differing only in their p53 status. The gene encoding mitochondrial ferredoxin reductase (protein, FR; gene, FDXR) was one of the few genes significantly induced by p53 after 5-FU treatment. The FR protein was localized to mitochondria and suppressed the growth of colon cancer cells when over-expressed. Targeted disruption of the FDXR gene in human colon cancer cells showed that it was essential for viability, and partial disruption of the gene resulted in decreased sensitivity to 5-FU-induced apoptosis. These data, coupled with the effects of pharmacologic inhibitors of reactive oxygen species, indicate that FR contributes to p53-mediated apoptosis through the generation of oxidative stress in mitochondria.

Antimetabolites, Antineoplastic↗