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PubMed1999

NF kappa B activity and target gene expression in the rat brain after one and two exposures to ionizing radiation.

The central nervous system injury that can result after radiotherapy has been suggested to involve induced gene expression and cytokine production. We have previously shown that irradiation of primary cultures of rat astrocytes results in the activation of NF kappa B. To determine whether such an effect also occurs in vivo, NF kappa B activity was analyzed in the cerebral cortex of the rat brain after whole body irradiation. After a single dose of 15 Gy, NF kappa B activity was increased by 2 h postirradiation, returning to unirradiated levels by 8 hours. The increase was dose-dependent beginning at 2 Gy and continuing to at least 22.5 Gy. NF kappa B activity in the irradiated cortex was not accompanied by I kappa B alpha degradation. When 7.5 Gy was delivered 24 h before the 15 Gy, the increase in NF kappa B activity after 15 Gy was significantly reduced. These results suggest that an initial exposure to radiation induced a refractory period in the brain during which the susceptibility of NF kappa B to activation by subsequent irradiation was significantly reduced. This period of reduced sensitivity to radiation was also apparent for the induction of the NF kappa B-regulated cytokines IL-1 beta, IL-6, and TNF alpha.

Animals↗
PubMedDate not supplied

The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse.

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.

Amino Acid Sequence↗
PubMed2004

Compatibility of column inlet and adsorbent designs for processing of corn endosperm extract by expanded bed adsorption.

Corn has emerged as a viable host for expression of recombinant proteins; targeted expression to the endosperm has received particular attention. The protein extracts from corn endosperm differ from those of traditional hosts in regard to the nature of residual solids and extracted matrix contaminants. Each of these differences presents reasons for considering expanded bed adsorption for product capture and new considerations for limitations of the method. In this work three inlet-flow distribution devices (mesh, glass ballotini, and localized mixing) and six adsorbents with different physical (size and density), chemical (ligand), and base matrix properties were evaluated to determine conditions compatible with processing of crude corn endosperm extract by expanded bed adsorption. Of the inlet devices evaluated, the design with localized mixing at the inlet (as produced commercially by UpFront Chromatography A/S, Copenhagen, DK) allowed solids up to 550 microm into the column without clogging for all flow rates evaluated. A mesh at the inlet with size restriction of either 50 microm or 80 microm became clogged with very small corn particles (< 44 microm). When glass ballotini was used, large particles (550 microm) passed through for high flow rates (570 cm/h), but even small (< 44 microm) particles became trapped at a lower flow rate (180 cm/h). The physical and chemical properties of the resin determined whether solids could be eluted. The denser UpFront adsorbents allowed for complete elution of larger and more concentrated corn solids than the currently available Amersham Streamline adsorbents (Amersham Biosciences, Piscataway, NJ) as a result of the former's higher flow rate for the desired 2x expansion (570 cm/h for UpFront vs. 180 cm/h for Streamline). All corn solids < 162 microm eluted through nonderivatized UpFront resin. Larger corn solids began to accumulate due to their elevated sedimentation velocities. Feeds of < 44 microm solids at 0.45% and 2.0% dry weight successfully eluted through ion exchange adsorbents (DEAE and SP) from UpFront. However, significant accumulation occurred when the solids size increased to a feed of < 96 microm solids, thus indicating a weak interaction between corn solids and both forms of ion exchange ligands. Expanded beds operated with Streamline ion exchange adsorbents (DEAE and SP) did not allow full elution of corn solids of < 44 microm. A hyperdiffuse style EBA resin produced by Biosepra (Ciphergen Biosystems, Fremont, CA) with CM functionality showed a severe interaction with corn solids that collapsed the expanded bed and could not be eliminated with elevated flow rates or higher salt concentration.

Adsorption↗
PubMed2003

Targeted transgene expression in rat brain using lentiviral vectors.

Direct gene transfer to the adult brain is dependent on vectors that transduce non-dividing cells, such as lentiviral vectors. Another aspect of the development of gene therapy to the brain is the need for cell-specific transgene expression. Expression from vesicular stomatitis virus G-protein (VSV-G) pseudotyped lentiviral vectors has been reported to be mainly neuron specific in the brain. We constructed cell-specific lentiviral vectors using the neuron-specific enolase (rNSE) or the glial fibrillary acidic protein (hGFAP) promoters and compared them to the ubiquitous human cytomegalovirus promoter (hCMV), a hybrid CMV/beta-actin promoter (CAG) and the promoter for human elongation factor 1 alpha (EF1 alpha). Our results showed that the hGFAP promoter was expressed only in glial cells, whereas rNSE was purely neuron specific, showing that VSV-G is pantropic in the rat striatum. We conclude that the VSV-G allows transduction of both glial and neuronal cells and the promoter dictates in what cell type the transgene will be expressed. The expression of transgenes exclusively in astrocytes would allow for local delivery of secreted transgene products, such as glial cell line-derived neurotrophic factor (GDNF), circumventing the anterograde transport that may induce unwanted side effects.

Analysis of Variance↗
PubMed2003

Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase.

Dopamine (DA) is the only catecholaminergic neurotransmitter in the fruit fly Drosophila melanogaster. Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo. In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH). The Drosophila TH gene (DTH) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal. We have performed ple rescue experiments with modified DTH transgenes. Our results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS. Based on this study, we generated a GAL4 driver transgene, TH-GAL4, containing regulatory sequences from the DTH 5' flanking and downstream coding regions. TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome. As a first application of this driver, we observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies. We propose that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila.

Animals↗
PubMed1999

Tyrosinase-related protein 2 promoter targets transgene expression to ocular and neural crest-derived tissues.

In an effort to identify a promoter suitable for studying early ocular development, we generated transgenic mice carrying the lacZ reporter gene linked to the tyrosinase-related protein 2 (TRP2) promoter. TRP2-lacZ was expressed in early retinal pigment epithelium (RPE) and early neural crest cells in embryos. The promoter activity was robust and consistent in independent transgenic lines. The transgene was also expressed in the optic nerve and neural crest-derived neuronal cells in which the endogenous TRP2 gene is not expressed. This suggests that repressor elements may be missing in the promoter used in this study. To test whether this promoter can be used to study melanocyte development, we cross-mated TRP2-lacZ transgenic mice with mice heterozygous for the Patch (Ph) mutation. The pattern of beta-galactosidase activity in the embryos correlates well with the pigmentation phenotype in postnatal and adult Ph/+ mice. We also generated transgenic mice expressing fibroblast growth factor 9 (FGF9) directed by the TRP2 promoter and examined the effect on ocular development. Ectopic expression of FGF9 in the early embryonic RPE switched its differentiation pathway to a neuronal fate, resulting in formation of a duplicated neural retina in transgenic mice. These studies demonstrate that the TRP2 promoter is valuable for transgenic studies of ocular differentiation and development of neural crest cells.

Animals↗
PubMed2001

Functional abrogation of p53 is required for T-Ag induced proliferation in cardiomyocytes.

Targeted expression of the SV40 large T-antigen oncoprotein (T-Ag) induces cardiomyocyte proliferation in the atria and ventricles of transgenic mice. Previous studies have identified the p53 tumor suppressor, p107 (a homologue of the retinoblastoma tumor suppressor), and p193 (a novel BH3 only proapoptosis protein) as prominent TAg binding proteins in cardiomyocyte cell lines derived from these transgenic mice. To further explore the significance of these protein-protein interactions in the regulation of cardiomyocyte proliferation, a transgene comprising the human atrial natriuretic factor (ANF) promoter and sequences encoding a mutant T-Ag lacking the p53 binding domain was generated. Repeated micro-injection of this DNA gave rise to genetically mosaic animals with minimal transgene content, suggesting that widespread cardiac expression of mutant T-Ag was deleterious. This notion was supported by the observation that the transgene was selectively lost from the cardiac myocytes (but not the cardiac fibroblasts) in the mosaic animals. Crosses between the mosaic mice and animals expressing a cardiac restricted dominant negative p53 resulted in transgene transmission with ensuing overt cardiac tumorigenesis. Transfection of the mutant T-Ag in embryonic stem (ES) cell-derived cardiomyocytes resulted in wide-spread cell death with characteristics typical of apoptosis. Co-transfection with a dominant negative p53 transgene rescued mutant TAg-induced cell death in the ES-derived cardiomyocyte cultures, resulting in a marked proliferative response similar to that seen in vivo with the rescued transgenic mouse study. These results indicate that T-Ag expression in the absence of p53 functional abrogation results in cardiomyocyte death.

Animals↗
PubMed1996

Targeted CNS expression of interferon-gamma in transgenic mice leads to hypomyelination, reactive gliosis, and abnormal cerebellar development.

Circumstantial and experimental evidence has implicated the immune cytokine interferon-gamma (IFN-gamma) as a key mediator in the pathological changes that are observed in many demyelinating disorders, including the most common human demyelinating disease, multiple sclerosis. To produce an animal model with which to study the effects of IFN-gamma on the CNS, we have generated transgenic mice in which the expression of IFN-gamma has been placed under the transcriptional control of the myelin basic protein (MBP) gene. Transgenic mice generated with this construct have a shaking/shivering phenotype that is similar to that observed in naturally occurring mouse models of hypomyelination (e.g., shiverer, jimpy, quaking), and these transgenic animals have dramatically less CNS myelin than control animals. Reactive gliosis and increased macrophage/microglial F4/80 immunostaining were also observed. Additionally, major histocompatibility complex (MHC) class I and class II mRNA levels were increased in the CNS of MBP/IFN-gamma transgenic mice, and the increase in MHC class I mRNA expression was detected in both white and gray matter regions. Furthermore, cerebellar granule cell migration was abnormal in these animals. These results strongly support the hypothesis that IFN-gamma is a key effector molecule in immune-mediated demyelinating disorders and indicate that the presence of this cytokine in the CNS may also disrupt the developing nervous system.

Animals↗
PubMed1996

Mitochondrial alterations induced by aspirin in rat hepatocytes expressing mitochondrially targeted green fluorescent protein (mtGFP).

Mitochondria in primary living hepatocytes were visualized in cells transfected with a chimeric plasmid encoding for the green fluorescent protein (GFP) of Aequorea victoria engineered to be specifically targeted to mitochondria, as described recently (Rizutto et al. (1995) Curr. Biol. 5, 635-642). The identification of the fluorescent organelles as authentic mitochondria was confirmed by double labeling with rhodamine 123. Acetylsalicylate treatment of hepatocytes induced in mitochondria typical morphological alterations closely analogous to the swelling promoted by acetylsalicylate in isolated mitochondria. Cyclosporin A, which in isolated mitochondria prevents the changes induced by acetylsalicylate, had no protective action but induced per se specific alterations in the morphology of mitochondria. Moreover, exposure of hepatocytes to cyclosporin A followed by acetylsalicylate caused the same mitochondrial changes induced by each of the two compounds separately. The structural alterations caused by acetylsalicylate were constantly associated with a decrease in mitochondrial urea synthesis and cell viability.

Animals↗
PubMed1990

Retrovirus vector-targeted inducible expression of human beta-interferon gene to B-cells.

We have introduced the human beta-interferon gene with its promoter region into murine B-cell and fibroblast cell lines via a Moloney murine leukemia virus (M-MuLV) vector and have studied the inducible expression of the beta-interferon gene as a function of the various retroviral vector designs. By deleting the enhancer within the 3' viral long terminal repeat (LTR), inserting the human beta-interferon gene, and varying placement of the immunoglobulin heavy chain enhancer, we were able to construct vectors which yielded proviruses with various cell type-specific regulation. One of the vectors (pT154) led to a greater than 21-fold increase in beta-interferon protein synthesis after viral infection in the two B-cell lines analyzed, while no inducibility was seen in the fibroblast cells. The data show that inducible beta-interferon expression within a MuLV vector was highly dependent on the absence of the viral enhancer region in the long terminal repeat and the orientation of the beta-interferon gene within the proviral transcriptional unit; the insertion of the immunoglobulin enhancer elevated both constitutive and (or) inducible expression of beta-interferon in B-cells but inhibited constitutive expression of this gene in fibroblasts.

Animals↗
PubMed1981

Noncodominant expression of target antigens recognized by human cytotoxic T lymphocytes.

Human CTL that recognized MHC-controlled determinants distinct from HLA-A. B, C, and D/DR antigens were tested in a family with nine siblings. Segregation analysis of positive CML reactions showed strong lysis of target cells of three HLA-identical siblings, inheriting the b and c MHC haplotypes (blc), but not of the parents or siblings inheriting only one of these haplotypes. Some cytotoxicity was seen against parental target cells, although it seemed to be qualitatively distinct from that directed against the b/c targets. Studies using the competitive inhibition technique showed that cells inheriting only the b or c haplotypes were not effective in decreasing the specific cytotoxicity. Furthermore, simultaneous inclusion of inhibitor cells of both the b and c haplotypes did not hinder the specific cytotoxicity. Induction of CTL recognizing this new determinant did not occur when either antigens of the b or c haplotypes were used as MLC stimulating cells, or when antigens of both haplotypes were presented simultaneously, but on separate stimulating cells, in a three-cell MLC. These results suggest that the target determinant recognized by these unusual CTL is complex; it may be formed through interactions of two surface molecules or by genetic complementation yielding a hybrid antigen.

Binding, Competitive↗
PubMed1996

Patterns of abnormal protein expression in target formations and unstructured cores.

Streaming of Z-disks and focal myofibrillar degeneration occur in target formations (TF) and unstructured cores (UC). Similar myofibrillar alterations are also part of the spectrum of ultrastructural reactions that can occur in the myopathies associated with myofibrillar degeneration and abnormal foci of desmin positivity. In the latter disorders, there is ectopic overexpression of dystrophin, neural cell adhesion molecule (NCAM), gelsolin, beta-amyloid precursor protein (beta APP) epitopes, alpha 1-antichymotrypsin (alpha 1-ACT), and many abnormal fiber regions are also strongly congophilic. Therefore, we searched for similar abnormalities in TF and UC. The UC and the center of TF show increased immunoreactivity for actin, alpha-actinin, gelsolin, dystrophin, beta APP epitopes, alpha 1-ACT, beta 2-microglobulin, desmin, and NCAM, but minimal or no congophilia. The periphery of the TF reacts strongly for nebulin but not for actin. The observed immunocytochemical alterations in TF and UC may represent a stereotyped cellular response associated with myofibrillar degeneration due to any cause. However, the three-dimensional profile of the TF and UC as well as their fiber-type specificity distinguish them from lesions that have similar immunocytochemical profiles in other myopathies.

Amyloid beta-Peptides↗
PubMed2006

Targeted MYCN expression affects cytotoxic potential of chemotherapeutic drugs in neuroblastoma cells.

Neuroblastoma (NB) is a solid childhood tumour that exhibits heterogeneous biological and clinical phenotypes. Multiple drug resistance marks a major complication especially in high-risk patients with advanced tumour stages and specific genetic aberrations, such as MYCN amplification and lp deletion. As an approach to further address the mechanisms of chemotherapeutic responsiveness of NB, we used a MYCN-inducible in vitro system and tested the susceptibility of NB cells to anti-tumour drugs currently included in NB treatment protocols dependent on MYCN expression. We observed cytotoxic effects using drug concentrations corresponding to blood plasma levels achieved in NB patients. The most potent drugs were microtubule inhibitors vindesin, paclitaxel and vincristin. Less effective were doxorubicine, arsenic trioxide, cisplatin, etoposide and carboplatin. Exposed to anti-tumour agents, NB cells with induced MYCN expression exhibited higher specific apoptosis than NB cells lacking MYCN expression. Anti-tumour drugs in MYCN-on cells accelerated G1-S phase transition, led to enhanced accumulation of cell populations in G2/M phase, and increased levels of apoptosis. In contrast, MYCN-off cell populations arrested in G1 and, to a smaller extent, in G2/M and exhibited delayed onset of apoptosis. In summary, apoptosis profiles and anti-proliferative potential of chemotherapeutic drugs, used at in vivo tolerable doses, are affected by MYCN overexpression and deregulated cell cycle in SH-EP(MYCN) cells.

Antineoplastic Agents↗
PubMed2003

Targeting connexin43 expression accelerates the rate of wound repair.

The repair of tissue damage is a key survival process in all organisms and involves the coordinated activation of several cell types. Cell-cell communication is clearly fundamental to this process, and a great deal is known about extracellular communication within the wound site via cytokines. Here we show that direct cell-cell communication through connexin 43 (Cx43) gap junction channels also plays a major role in the wound healing process. In two different wound healing models, incisional and excisional skin lesions, we show that a single topical application of Cx43 antisense gel brings about a transient downregulation of Cx43 protein levels, and this results in a dramatic increase in the rate of wound closure. Cx43 knockdown reduces inflammation, seen both macroscopically, as a reduction in swelling, redness, and wound gape, and microscopically, as a significant decrease in neutrophil numbers in the tissue around the wound. One long-term consequence of the improved rate of healing is a significant reduction in the extent of granulation tissue deposition and the subsequent formation of a smaller, less distorted, scar. This approach is likely to have widespread therapeutic applications in other injured tissues and opens up new avenues of research into improving the wound healing process.

Animals↗
PubMed2004

The promoter of brain-specific angiogenesis inhibitor 1-associated protein 4 drives developmentally targeted transgene expression mainly in adult cerebral cortex and hippocampus.

Restricting transgene expression to specific cell types and maintaining long-term expression are major goals for gene therapy. Previously, we cloned brain-specific angiogenesis inhibitor 1-associated protein 4 (BAI1-AP4), a novel brain-specific protein that interacts with BAI1, and found that it was developmentally upregulated in the adult brain. In this report, we isolated 5 kb of the 5' upstream sequence of the mouse BAI1-AP4 gene and analyzed its promoter activity. Functional analyses demonstrated that an Sp1 site was the enhancer, and the region containing the transcription initiation site and an AP2-binding site was the basal promoter. We examined the ability of the BAI1-AP4 promoter to drive adult brain-specific expression by using it to drive lacZ expression in transgenic (TG) mice. Northern blot analyses showed a unique pattern of beta-galactosidase expression in TG brain, peaking at 1 month after birth, like endogenous BAI1-AP4. Histological analyses demonstrated the same localization and developmental expression of beta-galactosidase and BAI1-AP4 in most neurons of the cerebral cortex and hippocampus. Our data indicate that TG mice carrying the BAI1-AP4 promoter could be a valuable model system for region-specific brain diseases.

Animals↗
PubMed2005

Toxoplasma gondii Hsp90 is a potential drug target whose expression and subcellular localization are developmentally regulated.

Two replicative forms characterize the asexual cycle of the protozoan parasite Toxoplasma gondii: rapidly growing tachyzoites and slowly dividing encysted bradyzoites. The mechanisms that regulate the transition between these two stages are not clearly understood. However, stress inducers that also activate heat shock protein expression can trigger formation of bradyzoites in vitro. Here, we studied the association of the T.gondii Hsp90 with modulation of parasite differentiation and response to stress stimuli using RH DeltaUPRT parasites and the cystogenic strain ME49 and a clone derivative of that strain, PK. Our results show that Hsp90 transcript and protein levels increase under stress or bradyzoite differentiation conditions. Moreover, fluorescence microscopy studies revealed that Hsp90 is present in the cytosol of tachyzoites and both in the nucleus and cytosol of mature bradyzoites, suggesting a correlation between its subcellular organization and these two developmental stages. To further characterize the role for Hsp90 in bradyzoite differentiation, T.gondii tachyzoite mutants that are defective in differentiation showed the same staining pattern as tachyzoites under differentiation conditions. In addition, geldanamycin, a benzoquinone ansamycin antibiotic capable of binding and disrupting the function of Hsp90, blocked conversion both from the tachyzoite to bradyzoite and the bradyzoite to tachyzoite stage, suggesting an essential role for this protein in the regulation of stage interconversion. These results thus suggest Hsp90 may play a role in stage switch.

Amino Acid Motifs↗