The niche-derived glial cell line-derived neurotrophic factor (GDNF induces migration of mouse spermatogonial stem/progenitor cells.

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Lisa Dovere

Full Text Available In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF, a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.

IB4(+) nociceptors mediate persistent muscle pain induced by GDNF.

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Alvarez, Pedro; Chen, Xiaojie; Bogen, Oliver; Green, Paul G; Levine, Jon D

2012-11-01

Skeletal muscle is a well-known source of glial cell line-derived neurotrophic factor (GDNF), which can produce mechanical hyperalgesia. Since some neuromuscular diseases are associated with both increased release of GDNF and intense muscle pain, we explored the role of GDNF as an endogenous mediator in muscle pain. Intramuscularly injected GDNF induced a dose-dependent (0.1-10 ng/20 μl) persistent (up to 3 wk) mechanical hyperalgesia in the rat. Once hyperalgesia subsided, injection of prostaglandin E(2) at the site induced a prolonged mechanical hyperalgesia (>72 h) compared with naïve rats (vibration increased muscle GDNF levels at 24 h, a time point where rats also exhibited marked muscle hyperalgesia. Intrathecal antisense oligodeoxynucleotides to mRNA encoding GFRα1, the canonical binding receptor for GDNF, reversibly inhibited eccentric exercise- and mechanical vibration-induced muscle hyperalgesia. Finally, electrophysiological recordings from nociceptors innervating the gastrocnemius muscle in anesthetized rats, revealed significant increase in response to sustained mechanical stimulation after local GDNF injection. In conclusion, these data indicate that GDNF plays a role as an endogenous mediator in acute and induction of chronic muscle pain, an effect likely to be produced by GDNF action at GFRα1 receptors located in IB4(+) nociceptors.

Crosstalk between VEGF-A/VEGFR2 and GDNF/RET signaling pathways

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Tufro, Alda; Teichman, Jason; Banu, Nazifa; Villegas, Guillermo

2007-01-01

Vascular endothelial growth factor (VEGF-A) plays multiple roles in kidney development: stimulates cell proliferation, survival, tubulogenesis, and branching morphogenesis. However, the mechanism that mediates VEGF-A induced ureteric bud branching is unclear. Glial-derived neurotrophic factor (GDNF) signaling through tyrosine kinase c-RET is the major regulator of ureteric bud branching. Here we examined whether VEGF-A regulates RET signaling. We determined that ureteric bud-derived cells express the main VEGF-A signaling receptor, VEGFR2 and RET, by RT-PCR, immunoblotting, and immunocytochemistry. We show that the VEGF-A isoform VEGF 165 induces RET-tyr 1062 phosphorylation in addition to VEGFR2 autophosphorylation, that VEGF 165 and GDNF have additive effects on RET-tyr 1062 phosphorylation, and that VEGFR2 and RET co-immunoprecipitate. Functionally, VEGF 165 induces ureteric bud cell proliferation and branching morphogenesis. Similarly, in embryonic kidney explants VEGF 165 induces RET-tyr 1062 phosphorylation and upregulates GDNF. These findings provide evidence for a novel cooperative interaction between VEGFR2 and RET that mediates VEGF-A functions in ureteric bud cells

Glial cell line-derived neurotrophic factor protects against high-fat diet-induced hepatic steatosis by suppressing hepatic PPAR-γ expression.

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Mwangi, Simon Musyoka; Peng, Sophia; Nezami, Behtash Ghazi; Thorn, Natalie; Farris, Alton B; Jain, Sanjay; Laroui, Hamed; Merlin, Didier; Anania, Frank; Srinivasan, Shanthi

2016-01-15

Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease.

In vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay

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Uchida, Aya; Kishi, Kasane; Aiyama, Yoshimi; Miura, Kento [Department of Veterinary Anatomy, The University of Tokyo, Yayoi, Tokyo, 113-8657 (Japan); Takase, Hinako M.; Suzuki, Hitomi; Kanai-Azuma, Masami [Department of Experimental Animal Model for Human Disease, Tokyo Medical and Dental University, Tokyo, 113-8510 (Japan); Iwamori, Tokuko [Center of Biomedical Research, Kyusyu University, Fukuoka, 812-8582 (Japan); Kurohmaru, Masamichi; Tsunekawa, Naoki [Department of Veterinary Anatomy, The University of Tokyo, Yayoi, Tokyo, 113-8657 (Japan); Kanai, Yoshiakira, E-mail: aykanai@mail.ecc.u-tokyo.ac.jp [Department of Veterinary Anatomy, The University of Tokyo, Yayoi, Tokyo, 113-8657 (Japan)

2016-08-05

In mouse testes, spermatogonial stem cells (SSCs), a subpopulation of GFRα1 (GDNF family receptor-α1)-positive spermatogonia, are widely distributed along the convoluted seminiferous tubules. The proliferation and differentiation of the SSCs are regulated in part by local expression of GDNF (glial cell-derived neurotorphic factor), one of major niche factors for SSCs. However, the in vivo dynamics of the GDNF-stimulated GFRα1-positive spermatogonia remains unclear. Here, we developed a simple method for transplanting DiI-labeled and GDNF-soaked beads into the mouse testicular interstitium. By using this method, we examined the dynamics of GFRα1-positive spermatogonia in the tubular walls close to the transplanted GDNF-soaked beads. The bead-derived GDNF signals were able to induce the stratified aggregate formation of GFRα1-positive undifferentiated spermatogonia by day 3 post-transplantation. Each aggregate consisted of tightly compacted A{sub single} and marginal A{sub paired}–A{sub aligned} GFRα1-positive spermatogonia and was surrounded by A{sub aligned} GFRα1-negative spermatogonia at more advanced stages. These data not only provide in vivo evidence for the inductive roles of GDNF in forming a rapid aggregation of GFRα1-positive spermatogonia but also indicate the usefulness of this in vivo assay system of various growth factors for the stem/progenitor spermatogonia in mammalian spermatogenesis. - Highlights: • A novel bead transplantation assay was developed to examine the in vivo effects of growth factors on spermatogonia. • A rapid aggregation of GFRα1-positive spermatogonia was induced by the transplanted GDNF-soaked beads. • Tightly-compacted A{sub single} and marginal A{sub paired}–A{sub aligned} spermatogonia were formed in each GFRα1-positive aggregate.

Cyclical and patch-like GDNF distribution along the basal surface of Sertoli cells in mouse and hamster testes.

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Takeshi Sato

Full Text Available BACKGROUND AND AIMS: In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs through GDNF family receptor α1 (GFRα1. It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules. CONCLUSION/SIGNIFICANCE: Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.

Role of glial cell line-derived neurotrophic factor (GDNF)-neural cell adhesion molecule (NCAM) interactions in induction of neurite outgrowth and identification of a binding site for NCAM in the heel region of GDNF

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Nielsen, Janne; Gotfryd, Kamil; Li, Shizhong

2009-01-01

NCAM-induced neurite outgrowth by being independent of NCAM polysialylation. Additionally, we investigated the structural basis for GDNF-NCAM interactions and find that NCAM Ig3 is necessary for GDNF binding. Furthermore, we identify within the heel region of GDNF a binding site for NCAM...

Expression dynamics of self-renewal factors for spermatogonial stem cells in the mouse testis.

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Sakai, Mizuki; Masaki, Kaito; Aiba, Shota; Tone, Masaaki; Takashima, Seiji

2018-04-16

Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells (SSCs). Although GDNF is indispensable for the maintenance of SSCs, the role of FGF2 in the testis remains to be elucidated. To clarify this, the expression dynamics and regulatory mechanisms of Fgf2 and Gdnf in the mouse testes were analyzed. It is well known that Sertoli cells express Gdnf, and its receptor is expressed in a subset of undifferentiated spermatogonia, including SSCs. However, we found that Fgf2 was mainly expressed in the germ cells and its receptors were expressed not only in the cultured spermatogonial cell line, but also in testicular somatic cells. Aging, hypophysectomy, retinoic acid treatment, and testicular injury induced distinct Fgf2 and Gdnf expression dynamics, suggesting a difference in the expression mechanism of Fgf2 and Gdnf in the testis. Such differences might cause a dynamic fluctuation of Gdnf/Fgf2 ratio depending on the intrinsic/extrinsic cues. Considering that FGF2-cultured spermatogonia exhibit more differentiated phenotype than those cultured with GDNF, FGF2 might play a role distinct from that of GDNF in the testis, despite the fact that both factors are self-renewal factor for SSC in vitro.

GDNF and neublastin protect against NMDA-induced excitotoxicity in hippocampal slice cultures

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Bonde, C; Kristensen, B W; Blaabjerg, M

2000-01-01

-producing HiB5 cells, were added to slice cultures I h before exposure to 10 microM NMDA for 48h. Neuronal cell death was monitored, before and during the NMDA exposure, by densitometric measurements of propidium iodide (PI) uptake and loss of Nissl staining. Both the addition of rhGDNF and NBN......The potential neuroprotective effects of glial cell line-derived neurotrophic factor (GDNF) and neublastin (NBN) against NMDA-induced excitotoxicity were examined in hippocampal brain slice cultures. Recombinant human GDNF (25-100 ng/ ml) or NBN, in medium conditioned by growth of transfected, NBN...

SorLA controls neurotrophic activity by sorting of GDNF and its receptors GFRα1 and RET

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Glerup, Simon; Lume, Maria; Olsen, Ditte

2013-01-01

is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic...

Mono-(2-ethylhexyl)-phthalate (MEHP) affects ERK-dependent GDNF signalling in mouse stem-progenitor spermatogonia

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Lucas, Benjamin E.G.; Fields, Christopher; Joshi, Neeraj; Hofmann, Marie-Claude

2012-01-01

Highlights: ► MEHP affects SSC proliferation in a dose- and time-dependent manner. ► MEHP does not increase apoptosis, necrosis or the production of ROS in SSCs. ► MEHP reduces the activity of the GDNF/ERK1/2/FOS signalling pathway in SSCs. ► MEHP does not affect the GDNF/SRC/MYCN signalling pathway in SSCs. -- Abstract: Many commercial and household products such as lubricants, cosmetics, plastics, and paint contain phthalates, in particular bis-(2-ethyhexyl)-phthalate (DEHP). As a consequence, phthalates have been found in a number of locations and foods (streambeds, household dust, bottled water and dairy products). Epidemiological and animal studies analysing phthalate exposure in males provide evidence of degradation in sperm quality, associated to an increase in the incidence of genital birth defects and testicular cancers. In the testis, spermatogenesis is maintained throughout life by a small number of spermatogonial stem cells (SSCs) that self-renew or differentiate to produce adequate numbers of spermatozoa. Disruption or alteration of SSC self-renewal induce decreased sperm count and sperm quality, or may potentially lead to testicular cancer. GDNF, or glial cell-line-derived neurotrophic factor, is a growth factor that is essential for the self-renewal of SSCs and continuous spermatogenesis. In the present study, the SSC-derived cell line C18-4 was used as a model for preliminary assessment of the effects of mono-(2-ethylhexyl)-phthalate (MEHP, main metabolite of DEHP) on spermatogonial stem cells. Our data demonstrate that MEHP disrupts one of the known GDNF signalling pathways in these cells. MEHP induced a decrease of C18-4 cell viability in a time- and dose-dependent manner, as well as a disruption of ERK1/2 activation but not of SRC signalling. As a result, we observed a decrease of expression of the transcription factor FOS, which is downstream of the GDNF/ERK1/2 axis in these cells. Taken together, our data suggest that MEHP exposure

Brain stem slice conditioned medium contains endogenous BDNF and GDNF that affect neural crest boundary cap cells in co-culture.

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Kaiser, Andreas; Kale, Ajay; Novozhilova, Ekaterina; Siratirakun, Piyaporn; Aquino, Jorge B; Thonabulsombat, Charoensri; Ernfors, Patrik; Olivius, Petri

2014-05-30

Conditioned medium (CM), made by collecting medium after a few days in cell culture and then re-using it to further stimulate other cells, is a known experimental concept since the 1950s. Our group has explored this technique to stimulate the performance of cells in culture in general, and to evaluate stem- and progenitor cell aptitude for auditory nerve repair enhancement in particular. As compared to other mediums, all primary endpoints in our published experimental settings have weighed in favor of conditioned culture medium, where we have shown that conditioned culture medium has a stimulatory effect on cell survival. In order to explore the reasons for this improved survival we set out to analyze the conditioned culture medium. We utilized ELISA kits to investigate whether brain stem (BS) slice CM contains any significant amounts of brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF). We further looked for a donor cell with progenitor characteristics that would be receptive to BDNF and GDNF. We chose the well-documented boundary cap (BC) progenitor cells to be tested in our in vitro co-culture setting together with cochlear nucleus (CN) of the BS. The results show that BS CM contains BDNF and GDNF and that survival of BC cells, as well as BC cell differentiation into neurons, were enhanced when BS CM were used. Altogether, we conclude that BC cells transplanted into a BDNF and GDNF rich environment could be suitable for treatment of a traumatized or degenerated auditory nerve. Copyright © 2014 Elsevier B.V. All rights reserved.

Protection by GDNF and other trophic factors against the dopamine-depleting effects of neurotoxic doses of methamphetamine.

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Cass, Wayne A; Peters, Laura E; Harned, Michael E; Seroogy, Kim B

2006-08-01

Repeated methamphetamine (METH) administration to animals can result in long-lasting decreases in striatal dopamine (DA) content. It has previously been shown that glial cell line-derived neurotrophic factor (GDNF) can reduce the DA-depleting effects of neurotoxic doses of METH. However, there are several other trophic factors that are protective against dopaminergic toxins. Thus, the present experiments further investigated the protective effect of GDNF as well as the protective effects of several other trophic factors. Male Fischer-344 rats were given an intracerebral injection of trophic factor (2-10 microg) 1 day before METH (5 mg/kg, s.c., 4 injections at 2-h intervals). Seven days later DA levels in the striatum were measured using high-performance liquid chromatography (HPLC). Initial experiments indicated that only intrastriatal GDNF, and not intranigral GDNF, was protective. Thereafter, all other trophic factors were administered into the striatum. Members of the GDNF family (GDNF, neurturin, and artemin) all provided significant protection against the DA-depleting effects of METH, with GDNF providing the greatest protection. Brain-derived neurotrophic factor, neurotrophin-3, acidic fibroblast growth factor, basic fibroblast growth factor, ciliary neurotrophic factor, transforming growth factor-alpha (TGF-alpha), heregulin beta1 (HRG-beta1), and amphiregulin (AR) provided no significant protection at the doses examined. These results suggest that the GDNF family of trophic factors can provide significant protection against the DA-depleting effects of neurotoxic doses of METH.

Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion

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Patwardhan Amol M

2005-01-01

Full Text Available Abstract Background Nerve growth factor (NGF, glial cell line-derived neurotrophic factor (GDNF and brain-derived neurotrophic factor (BDNF all play important roles in the development of the peripheral sensory nervous system. Additionally, these growth factors are proposed to modulate the properties of the sensory system in the adult under pathological conditions brought about by nerve injury or inflammation. We have examined the effects of NGF, GDNF and BDNF on adult rat trigeminal ganglion (TG neurons in culture to gain a better understanding of how these growth factors alter the cytochemical and functional phenotype of these neurons, with special attention to properties associated with nociception. Results Compared with no growth factor controls, GDNF, at 1 and 100 ng/ml, significantly increased by nearly 100% the number of neurons in culture at 5 days post-plating. A significant, positive, linear trend of increasing neuron number as a function of BDNF concentration was observed, also peaking at nearly 100%. NGF treatment was without effect. Chronic treatment with NGF and GDNF significantly and concentration-dependently increased 100 nM capsaicin (CAP-evoked calcitonin gene-related peptide (CGRP release, reaching approximately 300% at the highest concentration tested (100 ng/ml. Also, NGF and GDNF each augmented anandamide (AEA- and arachidonyl-2-chloroethylamide (ACEA-evoked CGRP release, while BDNF was without effect. Utilizing immunohistochemistry to account for the proportions of TRPV1- or CGRP-positive neurons under each growth factor treatment condition and then standardizing evoked CGRP release to these proportions, we observed that NGF was much more effective in enhancing CAP- and 50 mM K+-evoked CGRP release than was GDNF. Furthermore, NGF and GDNF each altered the concentration-response function for CAP- and AEA-evoked CGRP release, increasing the Emax without altering the EC50 for either compound. Conclusions Taken together, our

Pharmacokinetics and safety in rhesus monkeys of a monoclonal antibody-GDNF fusion protein for targeted blood-brain barrier delivery.

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Pardridge, William M; Boado, Ruben J

2009-10-01

Glial-derived neurotrophic factor (GDNF) is a potential therapy for stroke, Parkinson's disease, or drug addiction. However, GDNF does not cross the blood-brain barrier (BBB). GDNF is re-engineered as a fusion protein with a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR), which acts as a molecular Trojan horse to deliver the GDNF across the BBB. The pharmacokinetics (PK), toxicology, and safety pharmacology of the HIRMAb-GDNF fusion protein were investigated in Rhesus monkeys. The fusion protein was administered as an intravenous injection at doses up to 50 mg/kg over a 60 h period to 56 Rhesus monkeys. The plasma concentration of the HIRMAb-GDNF fusion protein was measured with a 2-site sandwich ELISA. No adverse events were observed in a 2-week terminal toxicology study, and no neuropathologic changes were observed. The PK analysis showed a linear relationship between plasma AUC and dose, a large systemic volume of distribution, as well as high clearance rates of 8-10 mL/kg/min. A no-observable-adverse-effect level is established in the Rhesus monkey for the acute administration of the HIRMAb-GDNF fusion protein. The fusion protein targeting the insulin receptor has a PK profile similar to a classical small molecule.

A monoclonal antibody-GDNF fusion protein is not neuroprotective and is associated with proliferative pancreatic lesions in parkinsonian monkeys.

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Sachiko Ohshima-Hosoyama

Full Text Available Glial cell line derived neurotrophic factor (GDNF is a neurotrophic factor that has neuroprotective effects in animal models of Parkinson's disease (PD and has been proposed as a PD therapy. GDNF does not cross the blood brain barrier (BBB, and requires direct intracerebral delivery to be effective. Trojan horse technology, in which GDNF is coupled to a monoclonal antibody (mAb against the human insulin receptor (HIR, has been proposed to allow GDNF BBB transport (ArmaGen Technologies Inc.. In this study we tested the feasibility of HIRMAb-GDNF to induce neuroprotection in parkinsonian monkeys, as well as its tolerability and safety. Adult rhesus macaques were assessed throughout the study with a clinical rating scale, a computerized fine motor skills task and general health evaluations. Following baseline measurements, the animals received a unilateral intracarotid artery MPTP injection. Seven days later the animals were evaluated, matched according to disability and blindly assigned to receive twice a week i.v. treatments (vehicle, 1 or 5 mg/kg HIRmAb-GDNF for a period of three months. HIRmAb-GDNF did not improve parkinsonian motor symptoms and induced a dose-dependent hypersensitivity reaction. Quantification of dopaminergic striatal optical density and stereological nigral cell counts did not demonstrate differences between treatment groups. Focal pancreatic acinar to ductular metaplasia (ADM was noted in four of seven animals treated with 1 mg/kg HIRmAb-GDNF; two of four with ADM also had focal pancreatic intraepithelial neoplasia 1B (PanIN-1B lesions. Minimal to mild, focal to multifocal, nonsuppurative myocarditis was noted in all animals in the 5 mg/kg treatment group. Our results demonstrate that HIRmAb-GDNF dosing in a monkey model of PD is not an effective neuroprotective strategy and may present serious health risks that should be considered when planning future use of the IR antibody as a carrier, or of any systemic treatment of a

Treatment with glial derived neurotropic factor (GDNF attenuates oxidative damages of spinal

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Tao Li

2016-05-01

Full Text Available Spinal cord injury (SCI is a serious and debilitating issue being suffered by wide population worldwide. Extensive treatment approaches have been tested and being verified for their efficacy. Owing to the nature of central nervous system (CNS, the resident stem cells would be triggered in response to any sort of trauma with nerve factors as their communication signals. Apart from physical injuries, damages due to oxidative stress also need to be addressed while CNS repair mechanism takes place. This study looks at the potential of glial derived nerve factor (GDNF in addressing the SCI in regard to oxidative damages. A total of 60 Wistar rats were clustered into five groups and GDNF at various concentrations was tested in each group. Assessments in terms of oxidative stress parameters were noted and analyzed accordingly. It was noted that GDNF had reduced oxidative damages and increased the levels of anti-oxidants in dose-dependent manner (p < 0.05. Though treatment with 10 mg/mL and 20 mg/mL showed significant changes as compared to control group, these treatment modalities remained insignificant among each other. In conclusion, we demonstrated that GDNF exerted a neuro-protective effect on CNS by inducing anti-oxidants and reducing the levels of oxidative stress in SCI induced rat models.

The glial cell line-derived neurotrophic factor (GDNF) does not acutely change acetylcholine release in developing and adult neuromuscular junction.

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Garcia, Neus; Santafé, Manel M; Tomàs, Marta; Lanuza, Maria A; Besalduch, Nuria; Priego, Merche; Tomàs, Josep

2010-08-16

We use immunocytochemistry to show that the trophic molecule glial cell line-derived neurotrophic factor (GDNF) and its receptor GDNF family receptor alpha-1 (GFRalpha-1) are present in both neonatal (P6) and adult (P45) rodent neuromuscular junctions (NMJ) colocalized with several synaptic markers. However, incubation with exogenous GDNF (10-200ng/ml, 1-3h), does not affect spontaneous ACh release. Moreover, GDNF does not change the size of the evoked ACh release from the weak and the strong axonal inputs on dually innervated postnatal endplates nor in the most developed singly-innervated synapses at P6 and P45. Our findings indicate that GDNF (unlike neurotrophins) does not acutely modulate transmitter release during the developmental process of synapse elimination nor as the NMJ matures. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Effects of combined BDNF and GDNF treatment on cultured dopaminergic midbrain neurons

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Sautter, J; Meyer, Morten; Spenger, C

1998-01-01

Neural transplantation is an experimental therapy for Parkinson's disease. Pretreatment of fetal donor tissue with neurotrophic factors may improve survival of grafted dopaminergic neurons. Free-floating roller tube cultures of fetal rat ventral mesencephalon were treated with brain-derived neuro......Neural transplantation is an experimental therapy for Parkinson's disease. Pretreatment of fetal donor tissue with neurotrophic factors may improve survival of grafted dopaminergic neurons. Free-floating roller tube cultures of fetal rat ventral mesencephalon were treated with brain......-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), or a combination of both. Dopamine content of the culture medium, the number of tyrosine hydroxylase-immunoreactive neurons, and culture volumes were moderately increased in the BDNF- and GDNF-treated cultures but significantly...... increased by 6.8-, 3.2- and 2.4-fold, respectively after treatment with the combination of both factors. We conclude that pretreatment of dopaminergic tissue in culture with a combination of BDNF and GDNF may be an effective means to improve the quality of tissue prior to grafting....

Optimal doses of EGF and GDNF act as biological response modifiers to improve porcine oocyte maturation and quality

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Valleh, Mehdi Vafaye; Zandi, Nahid Karimi; Rasmussen, Mikkel Aabech

2017-01-01

It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action of the opti......It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action...... of the optimum doses of EGF and GDNF on porcine oocyte maturation, porcine cumulus-oocyte complexes (COCs) were matured in defined porcine oocyte medium supplemented with EGF, GDNF or a combination of both at varying concentrations (0-100 ng/ml) for 44 h. Nuclear and cytoplasmic maturation were determined...

Nucleolin modulates the subcellular localization of GDNF-inducible zinc finger protein 1 and its roles in transcription and cell proliferation

International Nuclear Information System (INIS)

Dambara, Atsushi; Morinaga, Takatoshi; Fukuda, Naoyuki; Yamakawa, Yoshinori; Kato, Takuya; Enomoto, Atsushi; Asai, Naoya; Murakumo, Yoshiki; Matsuo, Seiichi; Takahashi, Masahide

2007-01-01

GZF1 is a zinc finger protein induced by glial cell-line-derived neurotrophic factor (GDNF). It is a sequence-specific transcriptional repressor with a BTB/POZ (Broad complex, Tramtrack, Bric a brac/Poxvirus and zinc finger) domain and ten zinc finger motifs. In the present study, we used immunoprecipitation and mass spectrometry to identify nucleolin as a GZF1-binding protein. Deletion analysis revealed that zinc finger motifs 1-4 of GZF1 mediate its association with nucleolin. When zinc fingers 1-4 were deleted from GZF1 or nucleolin expression was knocked down by short interference RNA (siRNA), nuclear localization of GZF1 was impaired. These results suggest that nucleolin is involved in the proper subcellular distribution of GZF1. In addition, overexpression of nucleolin moderately inhibited the transcriptional repressive activity of GZF1 whereas knockdown of nucleolin expression by siRNA enhanced its activity. Thus, the repressive activity of GZF1 is modulated by the level at which nucleolin is expressed. Finally, we found that knockdown of GZF1 and nucleolin expression markedly impaired cell proliferation. These findings suggest that the physiological functions of GZF1 may be regulated by the protein's association with nucleolin

Alterations in BDNF (brain derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor) serum levels in bipolar disorder: The role of lithium.

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Tunca, Zeliha; Ozerdem, Aysegul; Ceylan, Deniz; Yalçın, Yaprak; Can, Güneş; Resmi, Halil; Akan, Pınar; Ergör, Gül; Aydemir, Omer; Cengisiz, Cengiz; Kerim, Doyuran

2014-09-01

Brain-derived neurotrophic factor (BDNF) has been consistently reported to be decreased in mania or depression in bipolar disorders. Evidence suggests that Glial cell line-derived neurotrophic factor (GDNF) has a role in the pathogenesis of mood disorders. Whether GDNF and BDNF act in the same way across different episodes in bipolar disorders is unclear. BDNF and GDNF serum levels were measured simultaneously by enzyme-linked immunosorbent assay (ELISA) method in 96 patients diagnosed with bipolar disorder according to DSM-IV (37 euthymic, 33 manic, 26 depressed) in comparison to 61 healthy volunteers. SCID- I and SCID-non patient version were used for clinical evaluation of the patients and healthy volunteers respectively. Correlations between the two trophic factor levels, and medication dose, duration and serum levels of lithium or valproate were studied across different episodes of illness. Patients had significantly lower BDNF levels during mania and depression compared to euthymic patients and healthy controls. GDNF levels were not distinctive. However GDNF/BDNF ratio was higher in manic state compared to euthymia and healthy controls. Significant negative correlation was observed between BDNF and GDNF levels in euthymic patients. While BDNF levels correlated positively, GDNF levels correlated negatively with lithium levels. Regression analysis confirmed that lithium levels predicted only GDNF levels positively in mania, and negatively in euthymia. Small sample size in different episodes and drug-free patients was the limitation of thestudy. Current data suggests that lithium exerts its therapeutic action by an inverse effect on BDNF and GDNF levels, possibly by up-regulating BDNF and down-regulating GDNF to achieve euthymia. Copyright © 2014 Elsevier B.V. All rights reserved.

Glial cell-derived neurotrophic factor alleviates sepsis-induced neuromuscular dysfunction by decreasing the expression of γ- and α7-nicotinic acetylcholine receptors in an experimental rat model of neuromyopathy.

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Wang, Xin; Min, Su; Xie, Fei; Yang, Jun; Li, Liang; Chen, Jingyuan

2018-02-05

Sepsis-induced neuromuscular dysfunction results from up-regulation of the expression of γ- and α7-nicotinic acetylcholine receptors (nAChR). Although glial cell derived neurotrophic factor (GDNF) has been implicated in repairing and supporting neurons, little is known about the effects of GDNF on demyelination of nerves in sepsis. In this study, we tested the hypothesis that GDNF could alleviate sepsis-induced neuromuscular dysfunction by decreasing the expression of γ- and α7-nAChR in an experimental rat model of neuromyopathy. Rats were randomly divided into a sham group and a sepsis group. Levels of inflammatory factors, muscle function, and nicotinic acetylcholine receptors were tested in rats after cecal ligation and puncture (CLP). At 24 h after CLP, GDNF was injected around the sciatic nerve of sepsis rats, cytokines were detected by enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining was used to detect the expression of nAChRs. GDNF and its downstream effector (Erk1/2 and GFR-α), neuregulin-1 (NRG-1) and γ- and α7-nAChR were measured using Western blot analysis. The expression of GDNF reached a minimum at 24 h after CLP. Compared with the sham group, the release of cytokines and the expression of γ- and α7-nAChR were significantly increased in the sepsis group. The administration of GDNF significantly alleviated sepsis-induced neuromuscular dysfunction, as well as reducing the expression of γ- and α7-nAChR. In addition, the expression of Erk1/2, GFR-α, NRG-1 were significantly increased after GDNF treatment. GDNF administration may improve patient outcomes by reducing the demyelination of nerves and the expression of γ- and α7-nAChR. Copyright © 2018. Published by Elsevier Inc.

GDNF family ligands display distinct action profiles on cultured GABAergic and serotonergic neurons of rat ventral mesencephalon

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Ducray, Angélique; Krebs, Sandra H:; Schaller, Benoft

2006-01-01

Glial-cell-line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN), known as the GDNF family ligands (GFLs), influence the development, survival and differentiation of cultured dopaminergic neurons from ventral mesencephalon (VM). Detailed knowledge about...... factors for VM GABAergic and serotonergic neurons, demonstrating characteristic individual action profiles emphasizing their important and distinct roles during brain development....

Stressed and depressed? Check your GDNF for epigenetic repression.

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Miller, Courtney A

2011-01-27

Some adults fail to adapt to chronic stress, developing symptoms of depression and anxiety. In this issue of Neuron, Uchida and colleagues link maladaptive stress responses to GDNF through a comprehensive investigation of the neurotrophic factor's regulation. Further, this study is an excellent example for investigators interested in neuroepigenetics research. © 2011 Elsevier Inc. All rights reserved.

GDNF family ligands display distinct action profiles on cultured GABAergic and serotonergic neurons of rat ventral mesencephalon

DEFF Research Database (Denmark)

Ducray, Angélique; Krebs, Sandra H:; Schaller, Benoft

2006-01-01

the effects of GFLs on other neuronal populations in the VM is essential for their potential application as therapeutic molecules for Parkinson's disease. Hence, in a comparative study, we investigated the effects of GFLs on cell densities and morphological differentiation of gamma-aminobutyric acid......Glial-cell-line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN), known as the GDNF family ligands (GFLs), influence the development, survival and differentiation of cultured dopaminergic neurons from ventral mesencephalon (VM). Detailed knowledge about......-immunoreactive (GABA-ir) and serotonin-ir (5-HT-ir) neurons in primary cultures of E14 rat VM. We observed that all GFLs [10 ng/ml] significantly increased GABA-ir cell densities (1.6-fold) as well as neurite length/neuron. However, only GDNF significantly increased the number of primary neurites/neuron, and none...

Effects of cavernous nerve reconstruction on expression of nitric oxide synthase isoforms in rats.

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Schlenker, Boris; Matiasek, Kaspar; Saur, Dieter; Gratzke, Christian; Bauer, Ricarda M; Herouy, Yared; Arndt, Christian; Blesch, Armin; Hartung, Rudolf; Stief, Christian G; Weidner, Norbert; May, Florian

2010-12-01

To evaluate the expression of nitric oxide synthase (NOS) isoforms after various reconstruction techniques in rats, to improve the understanding of neuronal repair mechanisms after radical prostatectomy, as Schwann cell-seeded guidance tubes have been shown to promote cavernous nerve regeneration, and glial cell-line-derived neurotrophic factor (GDNF)-overexpressing Schwann cells enhance nerve regenerative capacity. Segments (5 mm) of the cavernous nerve were excised bilaterally, followed by immediate bilateral microsurgical reconstruction. In four rats per group, the eight nerves were reconstructed by autologous nerve grafting (A), interposition of Schwann cell-seeded silicon tubes (B), or silicon tubes seeded with GDNF-hypersecreting Schwann cells (C). Further rats were either sham-operated (D) or had nerve excision without repair (E). Erectile function was evaluated after 6 weeks by re-laparotomy, electrical nerve stimulation and morphological evaluation of reconstructed nerves. NOS isoform mRNA expression was analysed by reverse transcription-polymerase chain reaction in tissue specimens taken from the corpora cavernosa. GDNF-transduced Schwann cell grafts restored erectile function better than untransduced Schwann cell and autologous nerve grafts (88% vs 75% vs 38%; not significant). Tissue specimens in group C had the highest expression of neuronal NOS mRNA in relation to the neuronal marker PGP9.5 among all treatment groups (not significant). Compared to nerve grafts (A) and negative controls (E) nNOS/PGP9.5 expression was significantly higher (P Schwann cell grafts (P < 0.05). Restoration of erectile function is paralleled by an increase of neuronal NOS expression in rats. Further experiments will determine the physiological role of neuronal NOS in erectile nerve repair processes. © 2010 THE AUTHORS. JOURNAL COMPILATION © 2010 BJU INTERNATIONAL.

Systemic injection of AAV9-GDNF provides modest functional improvements in the SOD1G93A ALS rat but has adverse side effects.

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Thomsen, G M; Alkaslasi, M; Vit, J-P; Lawless, G; Godoy, M; Gowing, G; Shelest, O; Svendsen, C N

2017-04-01

Injecting proteins into the central nervous system that stimulate neuronal growth can lead to beneficial effects in animal models of disease. In particular, glial cell line-derived neurotrophic factor (GDNF) has shown promise in animal and cell models of Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS). Here, systemic AAV9-GDNF was delivered via tail vein injections to young rats to determine whether this could be a safe and functional strategy to treat the SOD1 G93A rat model of ALS and, therefore, translated to a therapy for ALS patients. We found that GDNF administration in this manner resulted in modest functional improvement, whereby grip strength was maintained for longer and the onset of forelimb paralysis was delayed compared to non-treated rats. This did not, however, translate into an extension in survival. In addition, ALS rats receiving GDNF exhibited slower weight gain, reduced activity levels and decreased working memory. Collectively, these results confirm that caution should be applied when applying growth factors such as GDNF systemically to multiple tissues.

GDNF gene is associated with tourette syndrome in a family study.

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Huertas-Fernández, Ismael; Gómez-Garre, Pilar; Madruga-Garrido, Marcos; Bernal-Bernal, Inmaculada; Bonilla-Toribio, Marta; Martín-Rodríguez, Juan Francisco; Cáceres-Redondo, María Teresa; Vargas-González, Laura; Carrillo, Fátima; Pascual, Alberto; Tischfield, Jay A; King, Robert A; Heiman, Gary A; Mir, Pablo

2015-07-01

Tourette syndrome is a disorder characterized by persistent motor and vocal tics, and frequently accompanied by the comorbidities attention deficit hyperactivity disorder and obsessive-compulsive disorder. Impaired synaptic neurotransmission has been implicated in its pathogenesis. Our aim was to investigate the association of 28 candidate genes, including genes related to synaptic neurotransmission and neurotrophic factors, with Tourette syndrome. We genotyped 506 polymorphisms in a discovery cohort from the United States composed of 112 families and 47 unrelated singletons with Tourette syndrome (201 cases and 253 controls). Genes containing significant polymorphisms were imputed to fine-map the signal(s) to potential causal variants. Allelic analyses in Tourette syndrome cases were performed to check the role in attention deficit hyperactivity disorder and obsessive-compulsive disorder comorbidities. Target polymorphisms were further studied in a replication cohort from southern Spain composed of 37 families and three unrelated singletons (44 cases and 73 controls). The polymorphism rs3096140 in glial cell line-derived neurotrophic factor gene (GDNF) was significant in the discovery cohort after correction (P = 1.5 × 10(-4) ). No linkage disequilibrium was found between rs3096140 and other functional variants in the gene. We selected rs3096140 as target polymorphism, and the association was confirmed in the replication cohort (P = 0.01). No association with any comorbidity was found. As a conclusion, a common genetic variant in GDNF is associated with Tourette syndrome. A defect in the production of GDNF could compromise the survival of parvalbumin interneurons, thus altering the excitatory/inhibitory balance in the corticostriatal circuitry. Validation of this variant in other family cohorts is necessary. © 2015 International Parkinson and Movement Disorder Society.

GDNF Gene Is Associated With Tourette Syndrome in a Family Study

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Huertas-Fernández, Ismael; Gómez-Garre, Pilar; Madruga-Garrido, Marcos; Bernal-Bernal, Inmaculada; Bonilla-Toribio, Marta; Martín-Rodríguez, Juan Francisco; Cáceres-Redondo, María Teresa; Vargas-González, Laura; Carrillo, Fátima; Pascual, Alberto; Tischfield, Jay A.; King, Robert A.; Heiman, Gary A.; Mir, Pablo

2016-01-01

Background Tourette syndrome is a disorder characterized by persistent motor and vocal tics, and frequently accompanied by the comorbidities attention deficit hyperactivity disorder and obsessive-compulsive disorder. Impaired synaptic neurotransmission has been implicated in its pathogenesis. Our aim was to investigate the association of 28 candidate genes, including genes related to synaptic neurotransmission and neurotrophic factors, with Tourette syndrome. Methods We genotyped 506 polymorphisms in a discovery cohort from the United States composed of 112 families and 47 unrelated singletons with Tourette syndrome (201 cases and 253 controls). Genes containing significant polymorphisms were imputed to fine-map the signal(s) to potential causal variants. Allelic analyses in Tourette syndrome cases were performed to check the role in attention deficit hyperactivity disorder and obsessive-compulsive disorder comorbidities. Target polymorphisms were further studied in a replication cohort from southern Spain composed of 37 families and three unrelated singletons (44 cases and 73 controls). Results The polymorphism rs3096140 in glial cell line–derived neurotrophic factor gene (GDNF) was significant in the discovery cohort after correction (P = 1.5 × 10−4). No linkage disequilibrium was found between rs3096140 and other functional variants in the gene. We selected rs3096140 as target polymorphism, and the association was confirmed in the replication cohort (P = 0.01). No association with any comorbidity was found. Conclusions As a conclusion, a common genetic variant in GDNF is associated with Tourette syndrome. A defect in the production of GDNF could compromise the survival of parvalbumin interneurons, thus altering the excitatory/inhibitory balance in the corticostriatal circuitry. Validation of this variant in other family cohorts is necessary. PMID:26096985

Electrical muscle stimulation elevates intramuscular BDNF and GDNF mRNA following peripheral nerve injury and repair in rats.

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Willand, Michael P; Rosa, Elyse; Michalski, Bernadeta; Zhang, Jennifer J; Gordon, Tessa; Fahnestock, Margaret; Borschel, Gregory H

2016-10-15

Despite advances in surgery, patients with nerve injuries frequently have functional deficits. We previously demonstrated in a rat model that daily electrical muscle stimulation (EMS) following peripheral nerve injury and repair enhances reinnervation, detectable as early as two weeks post-injury. In this study, we explain the enhanced early reinnervation observed with electrical stimulation. In two groups of rats, the tibial nerve was transected and immediately repaired. Gastrocnemius muscles were implanted with intramuscular electrodes for sham or muscle stimulation. Muscles were stimulated daily, eliciting 600 contractions for one hour/day, repeated five days per week. Sixteen days following nerve injury, muscles were assessed for functional reinnervation by motor unit number estimation methods using electromyographic recording. In a separate cohort of rats, surgical and electrical stimulation procedures were identical but muscles and distal nerve stumps were harvested for molecular analysis. We observed that stimulated muscles had significantly higher motor unit number counts. Intramuscular levels of brain-derived and glial cell line-derived neurotrophic factor (BDNF and GDNF) mRNA were significantly upregulated in muscles that underwent daily electrical stimulation compared to those without stimulation. The corresponding levels of trophic factor mRNA within the distal stump were not different from one another, indicating that the intramuscular electrical stimulus does not modulate Schwann cell-derived trophic factor transcription. Stimulation over a three-month period maintained elevated muscle-derived GDNF but not BDNF mRNA. In conclusion, EMS elevates intramuscular trophic factor mRNA levels which may explain how EMS enhances neural regeneration following nerve injury. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Effects of GDNF pretreatment on function and survival of transplanted fetal ventral mesencephalic cells in the 6-OHDA rat model of Parkinson's disease

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Andereggen, Lukas; Meyer, Morten; Guzman, Raphael

2009-01-01

assessed at 9 weeks posttransplantation. However, a tendency for higher TH-ir fiber outgrowth from the transplants in the GDNF pretreated groups as compared to corresponding controls was observed. Furthermore, GDNF pretreatment showed a tendency for a higher number of GIRK2 positive neurons in the grafts...

Axon Guidance of Sympathetic Neurons to Cardiomyocytes by Glial Cell Line-Derived Neurotrophic Factor (GDNF)

NARCIS (Netherlands)

Miwa, Keiko; Lee, Jong-Kook; Takagishi, Yoshiko; Opthof, Tobias; Fu, Xianming; Hirabayashi, Masumi; Watabe, Kazuhiko; Jimbo, Yasuhiko; Kodama, Itsuo; Komuro, Issei

2013-01-01

Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal

Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons

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Jensen, Pia; Ducray, A D; Widmer, H R

2015-01-01

shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10days......, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells....... to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which...

Delayed administration of glial cell line-derived neurotrophic factor (GDNF) protects retinal ganglion cells in a pig model of acute retinal ischemia

DEFF Research Database (Denmark)

Kyhn, Maria Voss; Klassen, Henry; Johansson, Ulrica Englund

2009-01-01

electroretinography (mfERG), quantification of NeuN positive cells and evaluation of the degree of retinal perivasculitis and inflammation 6 weeks after the insult. In the post-injection eyes (days 14, 28 and 42), the ratios of the iN1 and the iP2 amplitudes were 0.10 (95% CI: 0.05-0.15) and 0.09 (95% CI: 0.......04-0.16) in eyes treated with blank microspheres, and 0.24 (95% CI: 0.18-0.32) and 0.23 (95% CI: 0.15-0.33) in eyes treated with GDNF microspheres. These differences were statistically significant (P eyes...... injected with GDNF microspheres compared to eyes injected with blank microspheres. In eyes injected with GDNF microspheres the ganglion cell count was 9.5/field (s.e.m.: 2.1, n = 8), in eyes injected with blank microspheres it was 3.5/field (s.e.m.: 1.2, n = 7). This difference was statistically...

High-frequency stimulation of the globus pallidus interna nucleus modulates GFRα1 gene expression in the basal ganglia.

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Ho, Duncun Xun Kiat; Tan, Yong Chee; Tan, Jiayi; Too, Heng Phon; Ng, Wai Hoe

2014-04-01

Deep brain stimulation (DBS) is an established therapy for movement disorders such as Parkinson's disease (PD). Although the efficacy of DBS is clear, its precise molecular mechanism remains unknown. The glial cell line derived factor (GDNF) family of ligands has been shown to confer neuroprotective effects on dopaminergic neurons, and putaminal infusion of GDNF have been investigated in PD patients with promising results. Despite the potential therapeutic role of GDNF in alleviating motor symptoms, there is no data on the effects of electrical stimulation on GDNF-family receptor (GFR) expression in the basal ganglia structures. Here, we report the effects of electrical stimulation on GFRα1 isoforms, particularly GFRα1a and GFRα1b. Wistar rats underwent 2 hours of high frequency stimulation (HFS) at the globus pallidus interna nucleus. A control group was subjected to a similar procedure but without stimulation. The HFS group, sacrificed 24 hours after treatment, had a threefold decrease in mRNA expression level of GFRα1b (p=0.037), but the expression level reverted to normal 72 hours after stimulation. Our preliminary data reveal the acute effects of HFS on splice isoforms of GFRα1, and suggest that HFS may modulate the splice isoforms of GFRα1a and GFRα1b to varying degrees. Going forward, elucidating the interactions between HFS and GFR may shed new insights into the complexity of GDNF signaling in the nervous system and lead to better design of clinical trials using these signaling pathways to halt disease progression in PD and other neurodegenerative diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

Low-Intensity Extracorporeal Shock Wave Therapy Enhances Brain-Derived Neurotrophic Factor Expression through PERK/ATF4 Signaling Pathway

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Bohan Wang

2017-02-01

Full Text Available Low-intensity extracorporeal shock wave therapy (Li-ESWT is used in the treatment of erectile dysfunction, but its mechanisms are not well understood. Previously, we found that Li-ESWT increased the expression of brain-derived neurotrophic factor (BDNF. Here we assessed the underlying signaling pathways in Schwann cells in vitro and in penis tissue in vivo after nerve injury. The result indicated that BDNF were significantly increased by the Li-ESWT after nerve injury, as well as the expression of BDNF in Schwann cells (SCs, RT4-D6P2T in vitro. Li-ESWT activated the protein kinase RNA-like endoplasmic reticulum (ER kinase (PERK pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α, and enhanced activating transcription factor 4 (ATF4 in an energy-dependent manner. In addition, GSK2656157—an inhibitor of PERK—effectively inhibited the effect of Li-ESWT on the phosphorylation of PERK, eIF2α, and the expression of ATF4. Furthermore, silencing ATF4 dramatically attenuated the effect of Li-ESWT on the expression of BDNF, but had no effect on hypoxia-inducible factor (HIF1α or glial cell-derived neurotrophic factor (GDNF in Schwann cells. In conclusion, our findings shed new light on the underlying mechanisms by which Li-ESWT may stimulate the expression of BDNF through activation of PERK/ATF4 signaling pathway. This information may help to refine the use of Li-ESWT to further improve its clinical efficacy.

Transplantation of bone marrow mononuclear cells modulates hippocampal expression of growth factors in chronically epileptic animals.

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Zanirati, Gabriele; Azevedo, Pamella Nunes; Marinowic, Daniel Rodrigo; Rodrigues, Felipe; de Oliveira Dias, Ana Christina; Venturin, Gianina Teribele; Greggio, Samuel; Simão, Fabrício; DaCosta, Jaderson Costa

2015-05-01

In previous studies, transplantation of bone marrow mononuclear cells (BMMCs) in epileptic animals has been found to be neuroprotective. However, the mechanism by which the BMMCs act remains unclear. We hypothesize that BMMCs may provide neuroprotection to the epileptic brain through trophic support. To test our hypothesis, we studied the temporal expression of neurotrophins after BMMC transplantation in the epileptic rat hippocampus. Chronically epileptic rats were intravenously transplanted with 1 × 10(7) BMMCs isolated from GFP transgenic mice. Expression levels of BDNF, GDNF, NGF, VEGF, and TGF-β1, and their receptors, were evaluated by ELISA and/or qRT-PCR analysis. Our data revealed increased protein expression of BDNF, GDNF, NGF, and VEGF and reduced levels of TGF-β1 in the hippocampus of transplanted epileptic animals. Additionally, an increase in the mRNA expression of BDNF, GDNF, and VEGF, a reduction in TGF-β1, and a decrease in mRNA levels of the TrkA and TGFR-β1 receptors were also observed. The gain provided by transplanted BMMCs in the epileptic brain may be related to the ability of these cells in modulating the network of neurotrophins and angiogenic signals. © 2015 John Wiley & Sons Ltd.

The involvement of BDNF, NGF and GDNF in aging and Alzheimer’s disease

OpenAIRE

Budni, Josiane; Bellettini-Santos, Tatiani; Mina, Francielle; Garcez, Michelle Lima; Zugno, Alexandra Ioppi

2015-01-01

Aging is a normal physiological process accompanied by cognitive decline. This aging process has been the primary risk factor for development of aging-related diseases such as Alzheimer’s disease (AD). Cognitive deficit is related to alterations of neurotrophic factors level such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF). These strong relationship between aging and AD is important to investigate the time which they...

A novel GLP-1/GIP dual agonist is more effective than liraglutide in reducing inflammation and enhancing GDNF release in the MPTP mouse model of Parkinson's disease.

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Yuan, Ziyue; Li, Dongfang; Feng, Peng; Xue, Guofang; Ji, Chenhui; Li, Guanglai; Hölscher, Christian

2017-10-05

Type 2 diabetes mellitus (T2DM) is one of the risk factors for Parkinson's disease (PD). Insulin desensitisation has been observed in the brains of patients, which may promote neurodegeneration. Incretins are a family of growth factors that can re-sensitise insulin signalling. We have previously shown that mimetics of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) have neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP) mouse model of PD. Recently, dual GLP-1/GIP receptor agonists have been developed. We therefore tested the novel dual agonist DA3-CH in comparison with the best GLP-1 analogue currently on the market, liraglutide (both drugs 25nmol/kg ip once-daily for 7 days) in the MPTP mouse model of PD (25mg/kg ip once-daily for 7 days). In the Rotarod and grip strength assessment, DA3-CH was superior to liraglutide in reversing the MPTP-induced motor impairment. Dopamine synthesis as indicated by levels of tyrosine hydroxylase was much reduced by MPTP in the substantia nigra and striatum, and DA3-CH reversed this while liragutide only partially reversed this. The chronic inflammation response as shown in increased levels of activated microglia and astrocytes was reduced by both drugs. Importantly, expression levels of the neuroprotective growth factor Glial Derived Neurotrophic Factor (GDNF) was much enhanced by both DA3-CH and liragutide. The results demonstrate that the combination of GLP-1 and GIP receptor activation is superior to single GLP-1 receptor activation alone. Therefore, new dual agonists may be a promising treatment for PD. The GLP-1 receptor agonist exendin-4 has already shown disease modifying effects in clinical trials in PD patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancing Peripheral Nerve Regeneration with a Novel Drug Delivering Nerve Conduit

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2017-12-01

In Vitro NGF/GNDF release kinetics experiments .......................... (Gale, Agarwal) (11-18months) 2. ELISA detection of NGF/GDNF... ELISA for NGF/GDNF detection of day 30 animals ........... (Gale) (24-29 months) b. Walking Track...26-30months) a. ELISA for NGF/GDNF detection of day 30 animals ........... (Gale) (29-33 months) b. Walking Track

Transport of Glial Cell Line-Derived Neurotrophic Factor into Liposomes across the Blood-Brain Barrier: In Vitro and in Vivo Studies

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Shaoling Wu

2014-02-01

Full Text Available Glial cell line-derived neurotrophic factor (GDNF was encapsulated into liposomes in order to protect it from enzyme degradation in vivo and promote its permeability across the blood-brain barrier (BBB. In this study, GDNF conventional liposomes (GDNF-L and GDNF target sterically stabilized liposomes (GDNF-SSL-T were prepared. The average size of liposomes was below 90 nm. A primary model of BBB was established and evaluated by transendothelial electrical resistance (TEER and permeability. This BBB model was employed to study the permeability of GDNF liposomes in vitro. The results indicated that the liposomes could enhance transport of GDNF across the BBB and GDNF-SSL-T had achieved the best transport efficacy. The distribution of GDNF liposomes was studied in vivo. Free GDNF and GDNF-L were eliminated rapidly in the circulation. GDNF-SSL-T has a prolonged circulation time in the blood and favorable brain delivery. The values of the area under the curve (AUC(0–1 h in the brain of GDNF-SSL-T was 8.1 times and 6.8 times more than that of free GDNF and GDNF-L, respectively. These results showed that GDNF-SSL-T realized the aim of targeted delivery of therapeutic proteins to central nervous system.

Cabergoline decreases alcohol drinking and seeking behaviors via glial cell line-derived neurotrophic factor.

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Carnicella, Sebastien; Ahmadiantehrani, Somayeh; He, Dao-Yao; Nielsen, Carsten K; Bartlett, Selena E; Janak, Patricia H; Ron, Dorit

2009-07-15

Cabergoline is an ergotamine derivative that increases the expression of glial cell line-derived neurotrophic factor (GDNF) in vitro. We recently showed that GDNF in the ventral tegmental area (VTA) reduces the motivation to consume alcohol. We therefore set out to determine whether cabergoline administration decreases alcohol-drinking and -seeking behaviors via GDNF. Reverse transcription polymerase chain reaction (RT-PCR) and Enzyme-Linked ImmunoSorbent Assay (ELISA) were used to measure GDNF levels. Western blot analysis was used for phosphorylation experiments. Operant self-administration in rats and a two-bottle choice procedure in mice were used to assess alcohol-drinking behaviors. Instrumental performance tested during extinction was used to measure alcohol-seeking behavior. The [35S]GTPgammaS binding assay was used to assess the expression and function of the dopamine D2 receptor (D2R). We found that treatment of the dopaminergic-like cell line SH-SY5Y with cabergoline and systemic administration of cabergoline in rats resulted in an increase in GDNF level and in the activation of the GDNF pathway. Cabergoline treatment decreased alcohol-drinking and -seeking behaviors including relapse, and its action to reduce alcohol consumption was localized to the VTA. Finally, the increase in GDNF expression and the decrease in alcohol consumption by cabergoline were abolished in GDNF heterozygous knockout mice. Together, these findings suggest that cabergoline-mediated upregulation of the GDNF pathway attenuates alcohol-drinking behaviors and relapse. Alcohol abuse and addiction are devastating and costly problems worldwide. This study puts forward the possibility that cabergoline might be an effective treatment for these disorders.

Tissue-specific RNA expression marks distant-acting developmental enhancers.

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Han Wu

2014-09-01

Full Text Available Short non-coding transcripts can be transcribed from distant-acting transcriptional enhancer loci, but the prevalence of such enhancer RNAs (eRNAs within the transcriptome, and the association of eRNA expression with tissue-specific enhancer activity in vivo remain poorly understood. Here, we investigated the expression dynamics of tissue-specific non-coding RNAs in embryonic mouse tissues via deep RNA sequencing. Overall, approximately 80% of validated in vivo enhancers show tissue-specific RNA expression that correlates with tissue-specific enhancer activity. Globally, we identified thousands of tissue-specifically transcribed non-coding regions (TSTRs displaying various genomic hallmarks of bona fide enhancers. In transgenic mouse reporter assays, over half of tested TSTRs functioned as enhancers with reproducible activity in the predicted tissue. Together, our results demonstrate that tissue-specific eRNA expression is a common feature of in vivo enhancers, as well as a major source of extragenic transcription, and that eRNA expression signatures can be used to predict tissue-specific enhancers independent of known epigenomic enhancer marks.

New non-renal congenital disorders associated with medullary sponge kidney (MSK) support the pathogenic role of GDNF and point to the diagnosis of MSK in recurrent stone formers.

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Ria, Paolo; Fabris, Antonia; Dalla Gassa, Alessandra; Zaza, Gianluigi; Lupo, Antonio; Gambaro, Giovanni

2017-08-01

Medullary sponge kidney (MSK) is a congenital renal disorder. Its association with several developmental abnormalities in other organs hints at the likelihood of some shared step(s) in the embryogenesis of the kidney and other organs. It has been suggested that the REarranged during Transfection (RET) proto-oncogene and the Glial cell line-Derived Neurotrophic Factor (GDNF) gene are defective in patients with MSK, and both RET and GDNF are known to have a role in the development of the central nervous system, heart, and craniofacial skeleton. Among a cohort of 143 MSK patients being followed up for nephrolithiasis and chronic kidney disease at our institution, we found six with one or more associated non-renal anomalies: one patient probably has congenital hemihyperplasia and hypertrophic cardiomyopathy with adipose metaplasia and mitral valve prolapse; one has Marfan syndrome; and the other four have novel associations between MSK and nerve and skeleton abnormalities described here for the first time. The discovery of disorders involving the central nervous system, cardiovascular system and craniofacial skeleton in MSK patients supports the hypothesis of a genetic alteration on the RET-GDNF axis having a pivotal role in the pathogenesis of MSK, in a subset of patients at least. MSK seems more and more to be a systemic disease, and the identification of extrarenal developmental defects could be important in arousing the suspicion of MSK in recurrent stone formers.

Ageing of enteric neurons: oxidative stress, neurotrophic factors and antioxidant enzymes

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Korsak Kris

2012-08-01

Full Text Available Abstract Background Ageing is associated with gastrointestinal dysfunction, which can have a major impact on quality of life of the elderly. A number of changes in the innervation of the gut during ageing have been reported, including neuronal loss and degenerative changes. Evidence indicates that reactive oxygen species (ROS are elevated in ageing enteric neurons, but that neurotrophic factors may reduce generation of neuronal ROS. Two such factors, glial cell line derived neurotrophic factor (GDNF and neurotrophin-3 (NT-3 have also been found to protect enteric neurons against oxidative stress induced cell death of enteric ganglion cells in vitro. We have investigated the possible roles of neurotrophic factors further, by examining their expression in the gut during ageing, and by analysing their effects on antioxidant enzyme production in cultures of enteric ganglion cells. Results Analysis of the expression of GDNF and its receptors c-Ret and GFR α − 1 in rat gut by RT-PCR showed that expression continues throughout life and into ageing, in both ad libitum(AL and calorically-restricted (CR animals. Levels of expression of GDNF and GFR α − 1 were elevated in 24 month AL animals compared to 24 month CR animals, and to 24 CR and 6 month control animals respectively. The related factor Neurturin and its receptor GFR α − 2 were also expressed throughout life, the levels of the GFR – α-2(b isoform were reduced in 24 m AL animals. Immunolabelling showed that c-Ret and GFR α − 1 proteins were expressed by myenteric neurons in ageing animals. GDNF, but not NT-3, was found to increase expression of Cu/Zn superoxide dismutase and catalase by cultured enteric ganglion cells. Conclusions The neurotrophic factors GDNF and neurturin and their receptors continue to be expressed in the ageing gut. Changes in the levels of expression of GDNF , GFR α-1 and GFR α-2(b isoform occurred in 24 m AL animals. GDNF, but not

Urinary Levels of IL-1β and GDNF in Preterm Neonates as Potential Biomarkers of Motor Development: A Prospective Study

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Rafael Coelho Magalhães

2017-01-01

Full Text Available Objectives. To evaluate the association between inflammatory biomarkers, neurotrophic factors, birth conditions, and the presence of motor development abnormalities in preterm neonates. Methods. Plasma and urinary levels of cytokines (IL-1β, IL-6, IL-10, TNF, and IL-12p70, chemokines (CXCL8/IL-8, CCL2/MCP-1, CCL5/RANTES, CXCL10/IP-10, and CXCL9/MIG, and neurotrophic factors (BDNF and GDNF were evaluated in 40 preterm neonates born between 28 and 32 incomplete weeks of gestation, at four distinct time points: at birth (umbilical cord blood (T0, at 48 (T1, at 72 hours (T2, and at 3 weeks after birth (T3. Biomarkers levels were compared between different time points and then associated with Test of Infant Motor Performance (TIMP percentiles. Results. Maternal age, plasma, and urinary concentrations of inflammatory molecules and neurotrophic factors were significantly different between groups with normal versus lower than expected motor development. Higher levels of GDNF were found in the group with lower than expected motor development, while IL-1β and CXCL8/IL-8 values were higher in the group with typical motor development. Conclusion. Measurements of cytokines and neurotrophic factors in spot urine may be useful in the follow-up of motor development in preterm neonates.

Glial cell line-derived neurotrophic factor up-regulates GTP-cyclohydrolase I activity and tetrahydrobiopterin levels in primary dopaminergic neurones

DEFF Research Database (Denmark)

Bauer, M; Suppmann, S; Meyer, M

2002-01-01

in tetrahydrobiopterin levels whereas tyrosine 3-monooxygenase activity was not altered. Actinomycin D, asan inhibitor of de novo biosynthesis, abolished any GDNF-mediated up-regulation of GTPCH I activity. However, GTPCH I mRNA levels in primary dopaminergic neurones were not altered by GDNF treatment, suggesting...... by triggering activation of GTP-cyclohydrolase I (GTPCH I), a key enzyme in catecholamine biosynthesis. GDNF stimulation of primary dopaminergic neurones expressing both tyrosine 3-monooxygenase and GTPCH I resulted in a dose-dependent doubling of GTPCH I activity, and a concomitant increase...... that the mode of action for that up-regulation is not directly connected to the regulation of GTPCH I transcription. We conclude that GDNF, in addition to its action in structural differentiation, also promotes differentiation regarding expression and enzymatic activity of a crucial component...

NANOS2 acts downstream of glial cell line-derived neurotrophic factor signaling to suppress differentiation of spermatogonial stem cells.

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Sada, Aiko; Hasegawa, Kazuteru; Pin, Pui Han; Saga, Yumiko

2012-02-01

Stem cells are maintained by both stem cell-extrinsic niche signals and stem cell-intrinsic factors. During murine spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) signal emanated from Sertoli cells and germ cell-intrinsic factor NANOS2 represent key regulators for the maintenance of spermatogonial stem cells. However, it remains unclear how these factors intersect in stem cells to control their cellular state. Here, we show that GDNF signaling is essential to maintain NANOS2 expression, and overexpression of Nanos2 can alleviate the stem cell loss phenotype caused by the depletion of Gfra1, a receptor for GDNF. By using an inducible Cre-loxP system, we show that NANOS2 expression is downregulated upon the conditional knockout (cKO) of Gfra1, while ectopic expression of Nanos2 in GFRA1-negative spermatogonia does not induce de novo GFRA1 expression. Furthermore, overexpression of Nanos2 in the Gfra1-cKO testes prevents precocious differentiation of the Gfra1-knockout stem cells and partially rescues the stem cell loss phenotypes of Gfra1-deficient mice, indicating that the stem cell differentiation can be suppressed by NANOS2 even in the absence of GDNF signaling. Taken together, we suggest that NANOS2 acts downstream of GDNF signaling to maintain undifferentiated state of spermatogonial stem cells. Copyright © 2011 AlphaMed Press.

Identification of CHD1L as an Important Regulator for Spermatogonial Stem Cell Survival and Self-Renewal

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Shan-Shan Liu

2016-01-01

Full Text Available Chromodomain helicase/ATPase DNA binding protein 1-like gene (Chd1l participates in chromatin-dependent processes, including transcriptional activation and DNA repair. In this study, we have found for the first time that Chd1l is mainly expressed in the testicular tissues of prepubertal and adult mice and colocalized with PLZF, OCT4, and GFRα1 in the neonatal mouse testis and THY1+ undifferentiated spermatogonia or spermatogonial stem cells (SSCs. Knockdown of endogenous Chd1l in cultured mouse undifferentiated SSCs inhibited the expression levels of Oct4, Plzf, Gfrα1, and Pcna genes, suppressed SSC colony formation, and reduced BrdU incorporation, while increasing SSC apoptosis. Moreover, the Chd1l gene expression is activated by GDNF in the cultured mouse SSCs, and the GDNF signaling pathway was modulated by endogenous levels of Chd1l; as demonstrated by the gene expression levels of GDNF, inducible transcripts Etv5, Bcl6b, Pou3f, and Lhx1, but not that of GDNF-independent gene, Taf4b, were significantly downregulated by Chd1l knockdown in mouse SSCs. Taken together, this study provides the first evidence to support the notion that Chd1l is an intrinsic and novel regulator for SSC survival and self-renewal, and it exerts such regulation at least partially through a GDNF signaling pathway.

Intragenic HIV-1 env sequences that enhance gag expression

International Nuclear Information System (INIS)

Suptawiwat, Ornpreya; Sutthent, Ruengpung; Lee, T.-H.; Auewarakul, Prasert

2003-01-01

Expression of HIV-1 genes is regulated at multiple levels including the complex RNA splicing and transport mechanisms. Multiple cis-acting elements involved in these regulations have been previously identified in various regions of HIV-1 genome. Here we show that another cis-acting element was present in HIV-1 env region. This element enhanced the expression of Gag when inserted together with Rev response element (RRE) into a truncated HIV-1 genome in the presence of Rev. The enhancing activity was mapped to a 263-bp fragment in the gp41 region downstream to RRE. RNA analysis showed that it might function by promoting RNA stability and Rev-dependent RNA export. The enhancement was specific to Rev-dependent expression, since it did not enhance Gag expression driven by Sam68, a cellular protein that has been shown to be able to substitute for Rev in RNA export function

Targeting the Neural Microenvironment in Prostate Cancer

Science.gov (United States)

2017-10-01

neurotrophic factor (GDNF), which is expressed by peripheral nerves. GDNF binds to RET, a receptor tyrosine kinase, in conjunction with its co-receptor...in conjunction with its co- receptor GFRα1 and activates cellular signaling. Studies in pancreatic cancer strongly implicate RET signaling as a key

Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

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X. Joann You

2011-01-01

Full Text Available Work has shown that stem cell transplantation can rescue or replace neurons in models of retinal degenerative disease. Neural progenitor cells (NPCs modified to overexpress neurotrophic factors are one means of providing sustained delivery of therapeutic gene products in vivo. To develop a nonrodent animal model of this therapeutic strategy, we previously derived NPCs from the fetal cat brain (cNPCs. Here we use bicistronic feline lentiviral vectors to transduce cNPCs with glial cell-derived neurotrophic factor (GDNF together with a GFP reporter gene. Transduction efficacy is assessed, together with transgene expression level and stability during induction of cellular differentiation, together with the influence of GDNF transduction on growth and gene expression profile. We show that GDNF overexpressing cNPCs expand in vitro, coexpress GFP, and secrete high levels of GDNF protein—before and after differentiation—all qualities advantageous for use as a cell-based approach in feline models of neural degenerative disease.

Lesion-induced increase in survival and migration of human neural progenitor cells releasing GDNF

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Behrstock, Soshana; Ebert, Allison D.; Klein, Sandra; Schmitt, Melanie; Moore, Jeannette M.; Svendsen, Clive N.

2009-01-01

The use of human neural progenitor cells (hNPC) has been proposed to provide neuronal replacement or astrocytes delivering growth factors for brain disorders such as Parkinson’s and Huntington’s disease. Success in such studies likely requires migration from the site of transplantation and integration into host tissue in the face of ongoing damage. In the current study, hNPC modified to release glial cell line derived neurotrophic factor (hNPCGDNF) were transplanted into either intact or lesioned animals. GDNF release itself had no effect on the survival, migration or differentiation of the cells. The most robust migration and survival was found using a direct lesion of striatum (Huntington’s model) with indirect lesions of the dopamine system (Parkinson’s model) or intact animals showing successively less migration and survival. No lesion affected differentiation patterns. We conclude that the type of brain injury dictates migration and integration of hNPC which has important consequences when considering transplantation of these cells as a therapy for neurodegenerative diseases. PMID:19044202

Fibrin-Enhanced Canonical Wnt Signaling Directs Plasminogen Expression in Cementoblasts

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Saeed Ur Rahman

2017-11-01

Full Text Available Cementum is a mineralized layer on the tooth’s root surface and facilitates the biomechanical anchoring of fibrous connective tissues as a part of tooth-supportive complexes. Previously, we observed that OCCM30 cementoblasts cultured on fibrin matrices underwent apoptosis due to fibrin degradation through the expression of proteases. Here, we demonstrated that OCCM30 on fibrin matrices (OCCM30-fibrin enhanced canonical Wnt signaling, which directed to plasminogen expression. The OCCM30-fibrin showed higher levels of Wnt3a expression, nuclear translocation of β-catenin, and T-cell factor (TCF optimal motif (TOP reporter activity than the cells on tissue culture dishes (OCCM30-TCD, indicating that the OCCM30-fibrin enhanced canonical Wnt/β-catenin signaling. Also, OCCM30-fibrin expressed biomineralization-associated markers at higher levels than OCCM30-TCD, of which levels were further increased with LiCl, a Wnt signaling activator. The OCCM30 cementoblasts simultaneously showed that high levels of plasminogen, a critical component of fibrinolysis, were expressed in the OCCM30-fibrin. Activation of canonical Wnt signaling with LiCl treatment or with forced lymphoid enhancer factor 1 (LEF1-expression increased the expression of plasminogen. On the contrary, the inhibition of canonical Wnt signaling with siRNAs against Wnt3a or β-catenin abrogated fibrin-enhanced plasminogen expression. Furthermore, there are three conserved putative response elements for the LEF1/β-catenin complex in the plasminogen proximal promoter regions (−900 to +54. Site-directed mutations and chromatin immunoprecipitation indicated that canonical Wnt signaling directed plasminogen expression. Taken together, this study suggests that fibrin-based materials can modulate functional periodontal formations in controlling cementoblast differentiation and fibrin degradation.

Simultaneous Transplantation of Fetal Ventral Mesencephalic Tissue and Encapsulated Genetically Modified Cells Releasing GDNF in a Hemi-Parkinsonian Rat Model of Parkinson’s Disease

DEFF Research Database (Denmark)

Perez-Bouza, Alberto; Di Santo, Stefano; Seiler, Stefanie

2017-01-01

Transplantation of fetal ventral mesencephalic (VM) neurons for Parkinson's disease (PD) is limited by poor survival and suboptimal integration of grafted tissue into the host brain. In a 6-OHDA rat model of PD we investigated the feasibility of simultaneous transplantation of rat fetal VM tissue...... between groups were observed for the number of surviving TH-ir neurons or graft volume. In conclusion, our findings demonstrate that simultaneous transplantation of fetal VM tissue and encapsulated GDNF-releasing cells is feasible and support the graft survival and function. Pre-treatment of donor tissue...

Tenomodulin expression in the periodontal ligament enhances cellular adhesion.

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Yuske Komiyama

Full Text Available Tenomodulin (Tnmd is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.

Task-dependent enhancement of facial expression and identity representations in human cortex.

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Dobs, Katharina; Schultz, Johannes; Bülthoff, Isabelle; Gardner, Justin L

2018-05-15

What cortical mechanisms allow humans to easily discern the expression or identity of a face? Subjects detected changes in expression or identity of a stream of dynamic faces while we measured BOLD responses from topographically and functionally defined areas throughout the visual hierarchy. Responses in dorsal areas increased during the expression task, whereas responses in ventral areas increased during the identity task, consistent with previous studies. Similar to ventral areas, early visual areas showed increased activity during the identity task. If visual responses are weighted by perceptual mechanisms according to their magnitude, these increased responses would lead to improved attentional selection of the task-appropriate facial aspect. Alternatively, increased responses could be a signature of a sensitivity enhancement mechanism that improves representations of the attended facial aspect. Consistent with the latter sensitivity enhancement mechanism, attending to expression led to enhanced decoding of exemplars of expression both in early visual and dorsal areas relative to attending identity. Similarly, decoding identity exemplars when attending to identity was improved in dorsal and ventral areas. We conclude that attending to expression or identity of dynamic faces is associated with increased selectivity in representations consistent with sensitivity enhancement. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Growth enhancement and gene expression of Arabidopsis thaliana irradiated with active oxygen species

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Watanabe, Satoshi; Ono, Reoto; Hayashi, Nobuya; Shiratani, Masaharu; Tashiro, Kosuke; Kuhara, Satoru; Inoue, Asami; Yasuda, Kaori; Hagiwara, Hiroko

2016-07-01

The characteristics of plant growth enhancement effect and the mechanism of the enhancement induced by plasma irradiation are investigated using various active species in plasma. Active oxygen species in oxygen plasma are effective for growth enhancement of plants. DNA microarray analysis of Arabidopsis thaliana indicates that the genes coding proteins that counter oxidative stresses by eliminating active oxygen species are expressed at significantly high levels. The size of plant cells increases owing to oxygen plasma irradiation. The increases in gene expression levels and cell size suggest that the increase in the expression level of the expansin protein is essential for plant growth enhancement phenomena.

Measuring ability to enhance and suppress emotional expression: The Flexible Regulation of Emotional Expression (FREE) Scale.

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Burton, Charles L; Bonanno, George A

2016-08-01

Flexibility in self-regulatory behaviors has proved to be an important quality for adjusting to stressful life events and requires individuals to have a diverse repertoire of emotion regulation abilities. However, the most commonly used emotion regulation questionnaires assess frequency of behavior rather than ability, with little evidence linking these measures to observable capacity to enact a behavior. The aim of the current investigation was to develop and validate a Flexible Regulation of Emotional Expression (FREE) Scale that measures a person's ability to enhance and suppress displayed emotion across an array of hypothetical contexts. In Studies 1 and 2, a series of confirmatory factor analyses revealed that the FREE Scale consists of 4 first-order factors divided by regulation and emotional valence type that can contribute to 2 higher order factors: expressive enhancement ability and suppression ability. In Study 1, we also compared the FREE Scale to other commonly used emotion regulation measures, which revealed that suppression ability is conceptually distinct from suppression frequency. In Study 3, we compared the FREE Scale with a composite of traditional frequency-based indices of expressive regulation to predict performance in a previously validated emotional modulation paradigm. Participants' enhancement and suppression ability scores on the FREE Scale predicted their corresponding performance on the laboratory task, even when controlling for baseline expressiveness. These studies suggest that the FREE Scale is a valid and flexible measure of expressive regulation ability. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Simvastatin enhances bone morphogenetic protein receptor type II expression

International Nuclear Information System (INIS)

Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N.

2006-01-01

Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function

Enhanced subliminal emotional responses to dynamic facial expressions

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Wataru eSato

2014-09-01

Full Text Available Emotional processing without conscious awareness plays an important role in human social interaction. Several behavioral studies reported that subliminal presentation of photographs of emotional facial expressions induces unconscious emotional processing. However, it was difficult to elicit strong and robust effects using this method. We hypothesized that dynamic presentations of facial expressions would enhance subliminal emotional effects and tested this hypothesis with two experiments. Fearful or happy facial expressions were presented dynamically or statically in either the left or the right visual field for 20 (Experiment 1 and 30 (Experiment 2 ms. Nonsense target ideographs were then presented, and participants reported their preference for them. The results consistently showed that dynamic presentations of emotional facial expressions induced more evident emotional biases toward subsequent targets than did static ones. These results indicate that dynamic presentations of emotional facial expressions induce more evident unconscious emotional processing.

Differential tissue expression of enhanced green fluorescent protein in ‘Green mice’

OpenAIRE

Ma, De-Fu; Tezuka, Hideo; Kondo, Tetsuo; Sudo, Katsuko; Niu, Dong-Feng; Nakazawa, Tadao; Kawasaki, Tomonori; Yamane, Tetsu; Nakamura, Nobuki; Katoh, Ryohei

2010-01-01

In order to clarify tissue expression of enhanced green fluorescent protein (EGFP) in ‘green mice’ from a transgenic line having an EGFP cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer, we studied the expression of EGFP in various organs and tissues from these ‘green mice’ by immunohistochemistry with anti- EGFP antibody in conjunction with direct observation for EGFP fluorescence using confocal laser scanning microscopy. On i...

The Role of Neurotrophic Factors Conjugated to Iron Oxide Nanoparticles in Peripheral Nerve Regeneration: In Vitro Studies

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Ofra Ziv-Polat

2014-01-01

Full Text Available Local delivery of neurotrophic factors is a pillar of neural repair strategies in the peripheral nervous system. The main disadvantage of the free growth factors is their short half-life of few minutes. In order to prolong their activity, we have conjugated to iron oxide nanoparticles three neurotrophic factors: nerve growth factor (βNGF, glial cell-derived neurotrophic factor (GDNF, and basic fibroblast growth factor (FGF-2. Comparative stability studies of free versus conjugated factors revealed that the conjugated neurotrophic factors were significantly more stable in tissue cultures and in medium at 37°C. The biological effects of free versus conjugated neurotrophic factors were examined on organotypic dorsal root ganglion (DRG cultures performed in NVR-Gel, composed mainly of hyaluronic acid and laminin. Results revealed that the conjugated neurotrophic factors enhanced early nerve fiber sprouting compared to the corresponding free factors. The most meaningful result was that conjugated-GDNF, accelerated the onset and progression of myelin significantly earlier than the free GDNF and the other free and conjugated factors. This is probably due to the beneficial and long-acting effect that the stabilized conjugated-GDNF had on neurons and Schwann cells. These conclusive results make NVR-Gel enriched with conjugated-GDNF, a desirable scaffold for the reconstruction of severed peripheral nerve.

Prevention of Memory Impairment and Neurotrophic Factors Increased by Lithium in Wistar Rats Submitted to Pneumococcal Meningitis Model

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Lutiana R. Simões

2017-01-01

Full Text Available The aim of this study was to investigate the effects of lithium on brain-derived neurotrophic factor (BDNF, nerve growth factor (NGF, and glial cell line-derived neurotrophic factor (GDNF expression in the hippocampus and on memory in experimental pneumococcal meningitis. The mood-stabilizer lithium is known as a neuroprotective agent with many effects on the brain. In this study, animals received either artificial cerebrospinal fluid or Streptococcus pneumoniae suspension at a concentration of 5 × 109 CFU/mL. Eighteen hours after induction, all animals received ceftriaxone. The animals received saline or lithium (47.5 mg/kg or tamoxifen (1 mg/kg as adjuvant treatment, and they were separated into six groups: control/saline, control/lithium, control/tamoxifen, meningitis/saline, meningitis/lithium, and meningitis/tamoxifen. Ten days after meningitis induction, animals were subjected to open-field habituation and the step-down inhibitory avoidance tasks. Immediately after these tasks, the animals were killed and their hippocampus was removed to evaluate the expression of BDNF, NGF, and GDNF. In the meningitis group, treatment with lithium and tamoxifen resulted in improvement in memory. Meningitis group showed decreased expression of BDNF and GDNF in the hippocampus while lithium reestablished the neurotrophin expression. Lithium was able to prevent memory impairment and reestablishes hippocampal neurotrophin expression in experimental pneumococcal meningitis.

Hypoxia-inducible factor 1α regulates branching morphogenesis during kidney development.

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Tsuji, Kenji; Kitamura, Shinji; Makino, Hirofumi

2014-04-25

The kidneys are exposed to hypoxic conditions during development. Hypoxia-inducible factor (HIF), an important mediator of the response to hypoxia, is believed to have an important role in development. However, the relationship between HIF and branching morphogenesis has not been elucidated clearly. In this study, we examined whether HIF regulates kidney development. We harvested kidneys from day 13 rat embryos (E13Ks) and cultured the organs under normoxic (20% O2/5% CO2) or hypoxic (5% O2/5% CO2) conditions. We evaluated the kidneys based on morphology and gene expression. E13Ks cultured under hypoxic conditions had significantly more ureteric bud (UB) branching than the E13Ks cultured under normoxic conditions. In addition, the mRNA levels of GDNF and GDNF receptor (GFR-α1), increased under hypoxic conditions in E13Ks. When we cultured E13Ks with the HIF-1α inhibitor digoxin or with siRNA targeting HIF-1α under hypoxic conditions, we did not observe increased UB branching. In addition, the expression of GDNF and GFR-α1 was inhibited under hypoxic conditions when the kidneys were treated with siRNA targeting HIF-1α. We also elucidated that hypoxia inhibited UB cell apoptosis and promoted the expression of FGF7 mRNA levels in metanephric mesenchymal (MM) cells in vitro. These findings suggest that hypoxic condition has important roles in inducing branching morphogenesis during kidney development. Hypoxia might mediate branching morphogenesis via not only GDNF/Ret but also FGF signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase.

Science.gov (United States)

Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F

2010-10-08

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

Anodal tDCS targeting the right orbitofrontal cortex enhances facial expression recognition

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Murphy, Jillian M.; Ridley, Nicole J.; Vercammen, Ans

2015-01-01

The orbitofrontal cortex (OFC) has been implicated in the capacity to accurately recognise facial expressions. The aim of the current study was to determine if anodal transcranial direct current stimulation (tDCS) targeting the right OFC in healthy adults would enhance facial expression recognition, compared with a sham condition. Across two counterbalanced sessions of tDCS (i.e. anodal and sham), 20 undergraduate participants (18 female) completed a facial expression labelling task comprising angry, disgusted, fearful, happy, sad and neutral expressions, and a control (social judgement) task comprising the same expressions. Responses on the labelling task were scored for accuracy, median reaction time and overall efficiency (i.e. combined accuracy and reaction time). Anodal tDCS targeting the right OFC enhanced facial expression recognition, reflected in greater efficiency and speed of recognition across emotions, relative to the sham condition. In contrast, there was no effect of tDCS to responses on the control task. This is the first study to demonstrate that anodal tDCS targeting the right OFC boosts facial expression recognition. This finding provides a solid foundation for future research to examine the efficacy of this technique as a means to treat facial expression recognition deficits, particularly in individuals with OFC damage or dysfunction. PMID:25971602

Optimization of translation profiles enhances protein expression and solubility.

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Anne-Katrin Hess

Full Text Available mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

Structure-function correlation of chloroquine and analogues as transgene expression enhancers in nonviral gene delivery.

Science.gov (United States)

Cheng, Jianjun; Zeidan, Ryan; Mishra, Swaroop; Liu, Aijie; Pun, Suzie H; Kulkarni, Rajan P; Jensen, Gregory S; Bellocq, Nathalie C; Davis, Mark E

2006-11-02

To understand how chloroquine (CQ) enhances transgene expression in polycation-based, nonviral gene delivery systems, a number of CQ analogues with variations in the aliphatic amino side chain or in the aromatic ring are synthesized and investigated. Our studies indicate that the aliphatic amino moiety of CQ is essential to provide increased gene expression. Further, the enhancements are more dramatically affected by changes to the aromatic ring and are positively correlated to the strength of intercalation between DNA and the CQ analogues. Quinacrine (QC), a CQ analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to CQ at a concentration 10 times lower, while N(4)-(4-pyridinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CP), a CQ analogue that has a weakly intercalating pyridinyl ring, shows no effect on gene expression. Subtle change on the 7-substituent of the chloroquine aromatic structure can also greatly affect the ability of the CQ analogues to enhance transgene expression. Transfection in the presence of N(4)-(7-trifluoromethyl-4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at same concentration, while transfection in the presence of N(4)-(4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CQ7b) does not reveal any enhancing effects on expression. Through a number of comparative studies with CQ and its analogues, we conclude that there are at least three mechanistic features of CQ that lead to the enhancement in gene expression: (i) pH buffering in endocytic vesicles, (ii) displacement of polycations from the nucleic acids in polyplexes, and (iii) alteration of the biophysical properties of the released nucleic acid.

Differential tissue expression of enhanced green fluorescent protein in 'green mice'.

Science.gov (United States)

Ma, De-Fu; Tezuka, Hideo; Kondo, Tetsuo; Sudo, Katsuko; Niu, Dong-Feng; Nakazawa, Tadao; Kawasaki, Tomonori; Yamane, Tetsu; Nakamura, Nobuki; Katoh, Ryohei

2010-06-01

In order to clarify tissue expression of enhanced green fluorescent protein (EGFP) in 'green mice' from a transgenic line having an EGFP cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer, we studied the expression of EGFP in various organs and tissues from these 'green mice' by immunohistochemistry with anti- EGFP antibody in conjunction with direct observation for EGFP fluorescence using confocal laser scanning microscopy. On immunohistochemical examination and on direct observation by confocal laser scanning microscopy, the level of EGFP expression varied among organs and tissues. EGFP expression was diffusely and strongly observed in the skin, pituitary, thyroid gland, parathyroid gland, heart, gall bladder, pancreas, adrenals and urinary bladder. There was only sporadic and weak expression of EGFP in the epithelium of the trachea, bronchus of the lung, stratified squamous epithelium and gastric glands of the stomach, hepatic bile ducts of the liver, glomeruli and renal tubules of the kidney and endo-metrial glands of the uterus. Furthermore, EGFP was only demonstrated within the goblet and paneth cells in the colon and small intestine, the tall columnar cells in the ductus epididymis, and the leydig cells in the testis. In conclusion, our results show that EGFP is differentially expressed in organs and tissues of 'green mice', which indicates that 'green mice' may prove useful for research involving transplantation and tissue clonality.

Disruption of a -35kb enhancer impairs CTCF binding and MLH1 expression in colorectal cells.

Science.gov (United States)

Liu, Qing; Thoms, Julie A; Nunez, Andrea C; Huang, Yizhou; Knezevic, Kathy; Packham, Deborah; Poulos, Rebecca C; Williams, Rachel; Beck, Dominik; Hawkins, Nicholas J; Ward, Robyn L; Wong, Jason W H; Hesson, Luke B; Sloane, Mathew A; Pimanda, John

2018-06-13

MLH1 is a major tumour suppressor gene involved in the pathogenesis of Lynch syndrome and various sporadic cancers. Despite their potential pathogenic importance, genomic regions capable of regulating MLH1 expression over long distances have yet to be identified. Here we use chromosome conformation capture (3C) to screen a 650-kb region flanking the MLH1 locus to identify interactions between the MLH1 promoter and distal regions in MLH1 expressing and non-expressing cells. Putative enhancers were functionally validated using luciferase reporter assays, chromatin immunoprecipitation and CRISPR-Cas9 mediated deletion of endogenous regions. To evaluate whether germline variants in the enhancer might contribute to impaired MLH1 expression in patients with suspected Lynch syndrome, we also screened germline DNA from a cohort of 74 patients with no known coding mutations or epimutations at the MLH1 promoter. A 1.8kb DNA fragment, 35kb upstream of the MLH1 transcription start site enhances MLH1 gene expression in colorectal cells. The enhancer was bound by CTCF and CRISPR-Cas9 mediated deletion of a core binding region impairs endogenous MLH1 expression. 5.4% of suspected Lynch syndrome patients have a rare single nucleotide variant (G>A; rs143969848; 2.5% in gnomAD European, non-Finnish) within a highly conserved CTCF binding motif, which disrupts enhancer activity in SW620 colorectal carcinoma cells. A CTCF bound region within the MLH1 -35 enhancer regulates MLH1 expression in colorectal cells and is worthy of scrutiny in future genetic screening strategies for suspected Lynch syndrome associated with loss of MLH1 expression. Copyright ©2018, American Association for Cancer Research.

γ-Tocotrienol upregulates aryl hydrocarbon receptor expression and enhances the anticancer effect of baicalein

Energy Technology Data Exchange (ETDEWEB)

Yamashita, Shuya; Baba, Kiwako; Makio, Akiko; Kumazoe, Motofumi; Huang, Yuhui; Lin, I-Chian; Bae, Jaehoon; Murata, Motoki; Yamada, Shuhei; Tachibana, Hirofumi, E-mail: tatibana@agr.kyushu-u.ac.jp

2016-05-13

Previous studies have identified biomolecules that mediate the physiological actions of food factors, such as amino acids, vitamins, fatty acids, minerals, plant polyphenols, and lactobacilli, suggesting that our bodies are equipped with an innate system that senses which food factors are required to maintain our health. However, the effects of environmental factors on food factor sensing (FFS) remains largely unknown. Tocotorienols (T3s), which belongs to the vitamin E family, possess several physiological functions, including cholesterol lowering and neuroprotective effects. Here, we investigated the effects of naturally abundant γ-T3 on FFS-related gene expressions in melanoma using a DNA chip. Our results showed that γ-T3 increased the expression level of aryl hydrocarbon receptor (AhR), a sensing molecule to plant polyphenol baicalein. The co-treatment with γ-T3 and baicalein enhanced the anti-proliferative activity of baicalein, accompanied by the downstream events of AhR-activation induced by baicalein. These data suggest that γ-T3 upregulates AhR expression and enhances its sensitivity to baicalein. - Highlights: • γ-T3 upregulated the expression of AhR in mouse melanoma. • Promotion of the binding activity of Sp1 is associated with the increasing effect of γ-T3 on AhR expression. • γ-T3 enhanced the anti-proliferative activity of baicalein that has an AhR ligand activity. • γ-T3 enhanced the inducing activity of baicalein on the expression of AhR target genes.

Fibrin matrices with affinity-based delivery systems and neurotrophic factors promote functional nerve regeneration.

Science.gov (United States)

Wood, Matthew D; MacEwan, Matthew R; French, Alexander R; Moore, Amy M; Hunter, Daniel A; Mackinnon, Susan E; Moran, Daniel W; Borschel, Gregory H; Sakiyama-Elbert, Shelly E

2010-08-15

Glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity-based delivery of GDNF or NGF from fibrin-filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity-based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury.

Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability

OpenAIRE

Ruiz, Oscar N.; Alvarez, Derry; Torres, Cesar; Roman, Laura; Daniell, Henry

2011-01-01

Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) ...

Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ

International Nuclear Information System (INIS)

Kojima, Hiroyuki; Takeda, Yukimasa; Muromoto, Ryuta; Takahashi, Miki; Hirao, Toru; Takeuchi, Shinji; Jetten, Anton M.; Matsuda, Tadashi

2015-01-01

Highlights: • Nuclear receptors, RORα and RORγ, are key regulators of Th17 cell differentiation. • Isoflavones have RORα/γ agonistic activities. • Isoflavones enhance the interaction of RORα/γ with co-activator. • These compounds enhance the expression of Il17a mRNA in mouse EL4 cells. • Dietary isoflavones can act as modulators of Il17a expression via RORα/γ. - Abstract: The retinoic acid receptor-related orphan receptors α and γ (RORα and RORγ), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. In this study, we investigated the effects of isoflavones on RORα/γ activity and the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In doxycycline-inducible CHO stable cell lines, we found that four isoflavones, biochanin A (BA), genistein, formononetin, and daidzein, enhanced RORα- or RORγ-mediated transcriptional activity in a dose-dependent manner. In an activation assay of the Il17a promoter using Jurkat cells, these compounds enhanced the RORα- or RORγ-mediated activation of the Il17a promoter at concentrations of 1 × 10 −6 M to 1 × 10 −5 M. In mammalian two-hybrid assays, the four isoflavones enhanced the interaction between the RORα- or RORγ-ligand binding domain and the co-activator LXXLL peptide in a dose-dependent manner. In addition, these isoflavones potently enhanced Il17a mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin, but showed slight enhancement of Il17a gene expression in RORα/γ-knockdown EL4 cells. Immunoprecipitation and immunoblotting assays also revealed that BA enhanced the interaction between RORγt and SRC-1, which is a co-activator for nuclear receptors. Taken together, these results suggest that the isoflavones have the ability to enhance IL-17 gene expression by stabilizing the interactions between RORα/γ and co-activators. This also

Temporal profiles of age-dependent changes in cytokine mRNA expression and glial cell activation after status epilepticus in postnatal rat hippocampus.

Science.gov (United States)

Järvelä, Juha T; Lopez-Picon, Francisco R; Plysjuk, Anna; Ruohonen, Saku; Holopainen, Irma E

2011-04-08

Status epilepticus (SE) is proposed to lead to an age-dependent acute activation of a repertoire of inflammatory processes, which may contribute to neuronal damage in the hippocampus. The extent and temporal profiles of activation of these processes are well known in the adult brain, but less so in the developing brain. We have now further elucidated to what extent inflammation is activated by SE by investigating the acute expression of several cytokines and subacute glial reactivity in the postnatal rat hippocampus. SE was induced by an intraperitoneal (i.p.) injection of kainic acid (KA) in 9- and 21-day-old (P9 and P21) rats. The mRNA expression of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), matrix metalloproteinase-9 (MMP-9), glial-derived neurotrophic factor (GDNF), interferon gamma (IFN-γ), and transforming growth factor-beta 1 (TGF-β1) were measured from 4 h up to 3 days after KA injection with real-time quantitative PCR (qPCR). IL-1β protein expression was studied with ELISA, GFAP expression with western blotting, and microglial and astrocyte morphology with immunohistochemistry 3 days after SE. SE increased mRNA expression of IL-1β, TNF-α and IL-10 mRNA in hippocampus of both P9 and P21 rats, their induction being more rapid and pronounced in P21 than in P9 rats. MMP-9 expression was augmented similarly in both age groups and GDNF expression augmented only in P21 rats, whereas neither IFN-γ nor TGF-β1 expression was induced in either age group. Microglia and astrocytes exhibited activated morphology in the hippocampus of P21 rats, but not in P9 rats 3 d after SE. Microglial activation was most pronounced in the CA1 region and also detected in the basomedial amygdala. Our results suggest that SE provokes an age-specific cytokine expression in the acute phase, and age-specific glial cell activation in the subacute phase as verified now in the postnatal rat hippocampus. In the juvenile hippocampus

Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFα enhances GPR84 expression in adipocytes.

Science.gov (United States)

Nagasaki, Hiroshi; Kondo, Takaaki; Fuchigami, Masahiro; Hashimoto, Hiroyuki; Sugimura, Yoshihisa; Ozaki, Nobuaki; Arima, Hiroshi; Ota, Akira; Oiso, Yutaka; Hamada, Yoji

2012-02-17

In this study we aimed to identify the physiological roles of G protein-coupled receptor 84 (GPR84) in adipose tissue, together with medium-chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high-fat diet up-regulated GPR84 expression in fat pads. In 3T3-L1 adipocytes, co-culture with a macrophage cell line, RAW264, or TNFα remarkably enhanced GPR84 expression. In the presence of TNFα, MCFAs down-regulated adiponectin mRNA expression in 3T3-L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFα from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

Mechanical stimulation enhanced estrogen receptor expression and callus formation in diaphyseal long bone fracture healing in ovariectomy-induced osteoporotic rats.

Science.gov (United States)

Chow, S K H; Leung, K S; Qin, J; Guo, A; Sun, M; Qin, L; Cheung, W H

2016-10-01

Estrogen receptor (ER) in ovariectomy-induced osteoporotic fracture was reported to exhibit delayed expression. Mechanical stimulation enhanced ER-α expression in osteoporotic fracture callus at the tissue level. ER was also found to be required for the effectiveness of vibrational mechanical stimulation treatment in osteoporotic fracture healing. Estrogen receptor(ER) is involved in mechanical signal transduction in bone metabolism. Its expression was reported to be delayed in osteoporotic fracture healing. The purpose of this study was to investigate the roles played by ER during osteoporotic fracture healing enhanced with mechanical stimulation. Ovariectomy-induced osteoporotic SD rats that received closed femoral fractures were divided into five groups, (i) SHAM, (ii) SHAM-VT, (iii) OVX, (iv) OVX-VT, and (v) OVX-VT-ICI, where VT stands for whole-body vibration treatment and ICI for ER antagonization by ICI 182,780. Callus formation and gene expression were assessed at 2, 4, and 8 weeks postfracture. In vitro osteoblastic differentiation, mineralization, and ER-α expression were assessed. The delayed ER expression was found to be enhanced by vibration treatment. Callus formation enhancement was shown by callus morphometry and micro-CT analysis. Enhancement effects by vibration were partially abolished when ER was modulated by ICI 182,780, in terms of callus formation capacity at 2-4 weeks and ER gene and protein expression at all time points. In vitro, ER expression in osteoblasts was not enhanced by VT treatment, but osteoblastic differentiation and mineralization were enhanced under estrogen-deprived condition. When osteoblastic cells were modulated by ICI 182,780, enhancement effects of VT were eliminated. Vibration was able to enhance ER expression in ovariectomy-induced osteoporotic fracture healing. ER was essential in mechanical signal transduction and enhancement in callus formation effects during osteoporotic fracture healing enhanced by vibration

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase*

Science.gov (United States)

Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F.

2010-01-01

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr687 in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr687 and association with components of the Tyr1062 signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser696, a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr687 as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions. PMID:20682772

Correlation of contrast-enhanced ultrasound parameters with oncogene expression and cell proliferation activity in breast cancer

Institute of Scientific and Technical Information of China (English)

Ce Zhang

2017-01-01

Objective: To study the correlation of contrast-enhanced ultrasound parameters with oncogene expression and cell proliferation activity in breast cancer. Methods: Breast cancer lesions and benign breast lesions surgically removed in Zigong Third People's Hospital between May 2014 and February 2017 were selected, contrast-enhanced ultrasound was done before operation to draw the time-intensity curve and calculate the area under the curve (AUC), and the expression of proliferation molecules and tumor suppressor genes were detected after operation. Results:The contrast-enhanced ultrasound parameter AUC of the breast cancer lesion was greatly higher than that of the benign breast lesion; ECT2, ZKSCAN3, USP39 and EphA2 mRNA expression in breast cancer lesions were obviously higher than those in benign breast lesions whereas HPK1, TCEAL17, CCN5, ATG2B and ATG4D mRNA expression were greatly lower than those in benign breast lesions; ECT2, ZKSCAN3, USP39 and EphA2 mRNA expression in breast cancer lesions with high AUC were greatly higher than those in breast cancer lesions with low AUC whereas HPK1, TCEAL17, CCN5, ATG2B and ATG4D mRNA expression were greatly lower than those in breast cancer lesions with low AUC. Conclusion: The contrast-enhanced ultrasound parameter AUC of breast cancer lesion significantly increases and is closely related to the higher expression of pro-proliferation molecules and the lower expression of tumor suppressor genes.

Hydrogel Macroporosity and the Prolongation of Transgene Expression and the Enhancement of Angiogenesis

Science.gov (United States)

Shepard, Jaclyn A.; Virani, Farrukh R.; Goodman, Ashley G.; Gossett, Timothy D.; Shin, Seungjin; Shea, Lonnie D.

2012-01-01

The utility of hydrogels for regenerative medicine can be improved through localized gene delivery to enhance their bioactivity. However, current systems typically lead to low-level transgene expression located in host tissue surrounding the implant. Herein, we investigated the inclusion of macropores into hydrogels to facilitate cell ingrowth and enhance gene delivery within the macropores in vivo. Macropores were created within PEG hydrogels by gelation around gelatin microspheres, with gelatin subsequently dissolved by incubation at 37°C. The macropores were interconnected, as evidenced by homogeneous cell seeding in vitro and complete cell infiltration in vivo. Lentivirus loaded within hydrogels following gelation retained its activity relative to the unencapsulated control virus. In vivo, macroporous PEG demonstrated sustained, elevated levels of transgene expression for 6 weeks, while hydrogels without macropores had transient expression. Transduced cells were located throughout the macroporous structure, while non-macroporous PEG hydrogels had transduction only in the adjacent host tissue. Delivery of lentivirus encoding for VEGF increased vascularization relative to the control, with vessels throughout the macropores of the hydrogel. The inclusion of macropores within the hydrogel to enhance cell infiltration enhances transduction and influences tissue development, which has implications for multiple regenerative medicine applications. PMID:22800542

Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ

Science.gov (United States)

Kojima, Hiroyuki; Takeda, Yukimasa; Muromoto, Ryuta; Takahashi, Miki; Hirao, Toru; Takeuchi, Shinji; Jetten, Anton M.; Matsuda, Tadashi

2018-01-01

The retinoic acid receptor-related orphan receptors α and γ (RORα and RORγ), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. In this study, we investigated the effects of isoflavones on RORα/γ activity and the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In doxycycline-inducible CHO stable cell lines, we found that four isoflavones, biochanin A (BA), genistein, formononetin, and daidzein, enhanced RORα- or RORγ-mediated transcriptional activity in a dose-dependent manner. In an activation assay of the Il17a promoter using Jurkat cells, these compounds enhanced the RORα- or RORγ-mediated activation of the Il17a promoter at concentrations of 1 × 10−6 M to 1 × 10−5 M. In mammalian two-hybrid assays, the four isoflavones enhanced the interaction between the RORα- or RORγ-ligand binding domain and the co-activator LXXLL peptide in a dose-dependent manner. In addition, these isoflavones potently enhanced Il17a mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin, but showed slight enhancement of Il17a gene expression in RORα/γ-knockdown EL4 cells. Immunoprecipitation and immunoblotting assays also revealed that BA enhanced the interaction between RORγt and SRC-1, which is a co-activator for nuclear receptors. Taken together, these results suggest that the isoflavones have the ability to enhance IL-17 gene expression by stabilizing the interactions between RORα/γ and co-activators. This also provides the first evidence that dietary chemicals can enhance IL-17 gene expression in immune cells. PMID:25583575

Expressive Writing: Enhancing the Emotional Intelligence of Human Services Majors

Science.gov (United States)

Castillo, Yuleinys; Fischer, Jerome M.

2017-01-01

The skills and tasks in the human services field are highly connected to emotional intelligence abilities. The purpose of the study was to investigate the effect of an expressive writing program involving human service students in an undergraduate rehabilitation services course. The program was developed to enhance their emotional intelligence.…

Peripheral injury of pelvic visceral sensory nerves alters GFRa (GDNF family receptor alpha localization in sensory and autonomic pathways of the sacral spinal cord

Directory of Open Access Journals (Sweden)

Shelley Lynne Forrest

2015-04-01

Full Text Available GDNF (glial cell line-derived neurotrophic factor, neurturin and artemin use their co-receptors (GFRα1, GFRα2 and GFRα3, respectively and the tyrosine kinase Ret for downstream signalling. In rodent dorsal root ganglia (DRG most of the unmyelinated and some myelinated sensory afferents express at least one GFRα. The adult function of these receptors is not completely elucidated but their activity after peripheral nerve injury can facilitate peripheral and central axonal regeneration, recovery of sensation, and sensory hypersensitivity that contributes to pain. Our previous immunohistochemical studies of spinal cord and sciatic nerve injuries in adult rodents have identified characteristic changes in GFRα1, GFRα2 or GFRα3 in central spinal cord axons of sensory neurons located in dorsal root ganglia. Here we extend and contrast this analysis by studying injuries of the pelvic and hypogastric nerves that contain the majority of sensory axons projecting to the pelvic viscera (e.g., bladder and lower bowel. At 7 d, we detected some effects of pelvic but not hypogastric nerve transection on the ipsilateral spinal cord. In sacral (L6-S1 cord ipsilateral to nerve injury, GFRα1-immunoreactivity (IR was increased in medial dorsal horn and CGRP-IR was decreased in lateral dorsal horn. Pelvic nerve injury also upregulated GFRα1- and GFRα3-IR terminals and GFRα1-IR neuronal cell bodies in the sacral parasympathetic nucleus that provides the spinal parasympathetic preganglionic output to the pelvic nerve. This evidence suggests peripheral axotomy has different effects on somatic and visceral sensory input to the spinal cord, and identifies sensory-autonomic interactions as a possible site of post-injury regulation.

Multi-lipofection efficiently transfected genes into astrocytes in primary culture.

Science.gov (United States)

Wu, B Y; Liu, R Y; So, K L; Yu, A C

2000-10-30

This study demonstrated that liposome-mediated transfection - lipofection - is suitable for delivering genes into astrocytes. By repeatedly lipofecting the same astrocyte cultures, a process we call multi-lipofection, the transfection efficiency of the beta-galactosidase (beta-gal) gene was improved from 2.6+/-0.6 to 17. 4+/-1.1%. This is the highest efficiency ever reported in gene-transfer with Lipofectin(R) in a primary culture of mouse cerebral cortical astrocytes. Furthermore, multi-lipofection did not cause observable disturbance to astrocytes as indicated by insignificant changes in the glial fibrillary acidic protein content in the cultures. In order to demonstrate that the transfected gene achieved a physiologically relevant expression level, a plasmid containing the pEF-hsp70 protein gene was lipofected into astrocytes. This produced colonies of astrocytes showing an increased resistance to heat-induced cell death. A similar experiment was performed with the glial-derived neurotrophic factor (GDNF) gene. Control astrocytes had no detectable GDNF. In the transfected astrocytes, the GDNF protein could be identified intracellularly by immunocytochemistry. Western blot analysis revealed, as compared to astrocytes with one lipofection, a 2.9-fold increase of GDNF with four lipofections. GDNF remained detectable in astrocytes 2 weeks after four lipofections. Thus, multi-lipofection provides a mild and efficient means of delivering foreign genes into astrocytes in a primary culture, making astrocytes good candidate vehicle cells for gene/cell therapy in the CNS.

Association between smoking behaviour and genetic variants of ...

Indian Academy of Sciences (India)

ESZTER KOTYUK

Since the role of dopamine system in smoking is well established ... because increased GDNF levels attenuated the reward pro- cesses (Griffin ... methamphetamine self-administration, enhanced motivation ... the rewarding effects of cocaine.

Expression image data of Drosophila GAL4 enhancer trap lines - GETDB | LSDB Archive [Life Science Database Archive metadata

Lifescience Database Archive (English)

Full Text Available List Contact us GETDB Expression image data of Drosophila GAL4 enhancer trap lines Data detail Data name Exp...ta contents 3,075 expression image data by developmental stages of Drosophila Images are classified into the...escription Download License Update History of This Database Site Policy | Contact Us Expression image data of Drosophila GAL4 enhancer trap lines - GETDB | LSDB Archive ... ...ression image data of Drosophila GAL4 enhancer trap lines DOI 10.18908/lsdba.nbdc00236-004 Description of da

CRFR1 in the ventromedial caudate putamen modulates acute stress-enhanced expression of cocaine locomotor sensitization.

Science.gov (United States)

Liu, Shuli; Wang, Zhiyan; Li, Yijing; Sun, Xiaowei; Ge, Feifei; Yang, Mingda; Wang, Xinjuan; Wang, Na; Wang, Junkai; Cui, Cailian

2017-07-15

Repeated exposure to psychostimulants induces a long-lasting enhancement of locomotor activity called behavioral sensitization, which is often reinforced by stress after drug withdrawal. The mechanisms underlying these phenomena remain elusive. Here we explored the effects of acute stress 3 or 14 days after the cessation of chronic cocaine treatment on the expression of locomotor sensitization induced by a cocaine challenge in rats and the key brain region and molecular mechanism underlying the phenomenon. A single session of forced swimming, as an acute stress (administered 2 days after the cessation of cocaine), significantly enhanced the expression of cocaine locomotor sensitization 14 days after the final cocaine injection (challenge at 12 days after acute stress) but not 3 days after the cessation of cocaine (challenge at 1 day after acute stress). The result indicated that acute stress enhanced the expression of cocaine locomotor sensitization after incubation for 12 days rather than 1 day after the last cocaine injection. Moreover, the enhancement in locomotor sensitization was paralleled by a selective increase in the number of the c-Fos + cells, the level of CRFR1 mRNA in the ventromedial caudate putamen (vmCPu). Furthermore, the enhancement was significantly attenuated by CRFR1 antagonist NBI-27914 into the vmCPu, implying that the up-regulation of CRFR1 in the vmCPu seems to be critical in the acute stress-enhanced expression of cocaine locomotor sensitization. The findings demonstrate that the long-term effect of acute stress on the expression of cocaine locomotor sensitization is partially mediated by CRFR1 in the vmCPu. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells.

Science.gov (United States)

Hisaoka-Nakashima, Kazue; Miyano, Kanako; Matsumoto, Chie; Kajitani, Naoto; Abe, Hiromi; Okada-Tsuchioka, Mami; Yokoyama, Akinobu; Uezono, Yasuhito; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

2015-05-29

Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα(i/o) inhibitor, but not by NF449, a Gα(s) inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα(ο1) and Gα(i3) by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey(TM) assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα(i/o) activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα(i/o) upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

International Nuclear Information System (INIS)

Salem, Tamer Z.; Zhang, Fengrui; Thiem, Suzanne M.

2013-01-01

Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

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Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: smthiem@msu.edu [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)

2013-01-20

Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

In vitro efficacy of a gene-activated nerve guidance conduit incorporating non-viral PEI-pDNA nanoparticles carrying genes encoding for NGF, GDNF and c-Jun.

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Lackington, William A; Raftery, Rosanne M; O'Brien, Fergal J

2018-06-07

Despite the success of tissue engineered nerve guidance conduits (NGCs) for the treatment of small peripheral nerve injuries, autografts remain the clinical gold standard for larger injuries. The delivery of neurotrophic factors from conduits might enhance repair for more effective treatment of larger injuries but the efficacy of such systems is dependent on a safe, effective platform for controlled and localised therapeutic delivery. Gene therapy might offer an innovative approach to control the timing, release and level of neurotrophic factor production by directing cells to transiently sustain therapeutic protein production in situ. In this study, a gene-activated NGC was developed by incorporating non-viral polyethyleneimine-plasmid DNA (PEI-pDNA) nanoparticles (N/P 7 ratio, 2μg dose) with the pDNA encoding for nerve growth factor (NGF), glial derived neurotrophic factor (GDNF) or the transcription factor c-Jun. The physicochemical properties of PEI-pDNA nanoparticles, morphology, size and charge, were shown to be suitable for gene delivery and demonstrated high Schwann cell transfection efficiency (60±13%) in vitro. While all three genes showed therapeutic potential in terms of enhancing neurotrophic cytokine production while promoting neurite outgrowth, delivery of the gene encoding for c-Jun showed the greatest capacity to enhance regenerative cellular processes in vitro. Ultimately, this gene-activated NGC construct was shown to be capable of transfecting both Schwann cells (S42 cells) and neuronal cells (PC12 and dorsal root ganglia) in vitro, demonstrating potential for future therapeutic applications in vivo. The basic requirements of biomaterial-based nerve guidance conduits have now been well established and include being able to bridge a nerve injury to support macroscopic guidance between nerve stumps, while being strong enough to withstand longitudinal tension and circumferential compression, in addition to being mechanically sound to facilitate

Plasma glial cell line-derived neurotrophic factor in patients with major depressive disorder: a preliminary study.

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Lee, Bun-Hee; Hong, Jin-Pyo; Hwang, Jung-A; Na, Kyoung-Sae; Kim, Won-Joong; Trigo, Jose; Kim, Yong-Ku

2016-02-01

Some clinical studies have reported reduced peripheral glial cell line-derived neurotrophic factor (GDNF) level in elderly patients with major depressive disorder (MDD). We verified whether a reduction in plasma GDNF level was associated with MDD. Plasma GDNF level was measured in 23 healthy control subjects and 23 MDD patients before and after 6 weeks of treatment. Plasma GDNF level in MDD patients at baseline did not differ from that in healthy controls. Plasma GDNF in MDD patients did not differ significantly from baseline to the end of treatment. GDNF level was significantly lower in recurrent-episode MDD patients than in first-episode patients before and after treatment. Our findings revealed significantly lower plasma GDNF level in recurrent-episode MDD patients, although plasma GDNF levels in MDD patients and healthy controls did not differ significantly. The discrepancy between our study and previous studies might arise from differences in the recurrence of depression or the ages of the MDD patients.

Effect of adenovirus infection on transgene expression under the adenoviral MLP/TPL and the CMVie promoter/enhancer in CHO cells

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Mohamed A. El-Mogy

2017-06-01

Full Text Available The adenovirus major late promoter (MLP and its translational regulator – the tripartite leader (TPL sequence – can actively drive efficient gene expression during adenoviral infection. However, both elements have not been widely tested in transgene expression outside of the adenovirus genome context. In this study, we tested whether the combination of MLP and TPL would enhance transgene expression beyond that of the most widely used promoter in transgene expression in mammalian cells, the cytomegalovirus immediate early (CMVie promoter/enhancer. The activity of these two regulatory elements was compared in Chinese hamster ovary (CHO cells. Although transient expression was significantly higher under the control of the CMVie promoter/enhance compared to the MLP/TPL, this difference was greater at the level of transcription (30 folds than translation (11 folds. Even with adenovirus infection to provide additional elements (in trans, CMVie promoter/enhancer exhibited significantly higher activity relative to MLP/TPL. Interestingly, the CMVie promoter/enhancer was 1.9 folds more active in adenovirus-infected cells than in non-infected cells. Our study shows that the MLP-TPL drives lower transgene expression than the CMVie promoter/enhancer particularly at the transcription level. The data also highlight the utility of the TPL sequence at the translation level and/or possible overwhelming of the cellular translational machinery by the high transcription activity of the CMVie promoter/enhancer. In addition, here we present data that show stimulation of the CMVie promoter/enhancer by adenovirus infection, which may prove interesting in future work to test the combination of CMVie/TPL sequence, and additional adenovirus elements, for transgene expression.

HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression

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Redmond, Catherine J.; Dooley, Katharine E.; Fu, Haiqing; Gillison, Maura L.; Akagi, Keiko; Symer, David E.; Aladjem, Mirit I.

2018-01-01

Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted de novo super-enhancer formation. Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression. PMID:29364907

Quantitative automated microscopy (QuAM elucidates growth factor specific signalling in pain sensitization

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Levine Jon D

2010-12-01

Full Text Available Abstract Background Dorsal root ganglia (DRG-neurons are commonly characterized immunocytochemically. Cells are mostly grouped by the experimenter's eye as "marker-positive" and "marker-negative" according to their immunofluorescence intensity. Classification criteria remain largely undefined. Overcoming this shortfall, we established a quantitative automated microscopy (QuAM for a defined and multiparametric analysis of adherent heterogeneous primary neurons on a single cell base. The growth factors NGF, GDNF and EGF activate the MAP-kinase Erk1/2 via receptor tyrosine kinase signalling. NGF and GDNF are established factors in regeneration and sensitization of nociceptive neurons. If also the tissue regenerating growth factor, EGF, influences nociceptors is so far unknown. We asked, if EGF can act on nociceptors, and if QuAM can elucidate differences between NGF, GDNF and EGF induced Erk1/2 activation kinetics. Finally, we evaluated, if the investigation of one signalling component allows prediction of the behavioral response to a reagent not tested on nociceptors such as EGF. Results We established a software-based neuron identification, described quantitatively DRG-neuron heterogeneity and correlated measured sample sizes and corresponding assay sensitivity. Analysing more than 70,000 individual neurons we defined neuronal subgroups based on differential Erk1/2 activation status in sensory neurons. Baseline activity levels varied strongly already in untreated neurons. NGF and GDNF subgroup responsiveness correlated with their subgroup specificity on IB4(+- and IB4(--neurons, respectively. We confirmed expression of EGF-receptors in all sensory neurons. EGF treatment induced STAT3 translocation into the nucleus. Nevertheless, we could not detect any EGF induced Erk1/2 phosphorylation. Accordingly, intradermal injection of EGF resulted in a fundamentally different outcome than NGF/GDNF. EGF did not induce mechanical hyperalgesia, but blocked

Enhanced expression of Ang-(1-7 during pregnancy

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Brosnihan K.B.

2004-01-01

Full Text Available Pregnancy is a physiological condition characterized by a progressive increase of the different components of the renin-angiotensin system (RAS. The physiological consequences of the stimulated RAS in normal pregnancy are incompletely understood, and even less understood is the question of how this system may be altered and contribute to the hypertensive disorders of pregnancy. Findings from our group have provided novel insights into how the RAS may contribute to the physiological condition of pregnancy by showing that pregnancy increases the expression of both the vasodilator heptapeptide of the RAS, angiotensin-(1-7 [Ang-(1-7], and of a newly cloned angiotensin converting enzyme (ACE homolog, ACE2, that shows high catalytic efficiency for Ang II metabolism to Ang-(1-7. The discovery of ACE2 adds a new dimension to the complexity of the RAS by providing a new arm that may counter-regulate the activity of the vasoconstrictor component, while amplifying the vasodilator component. The studies reviewed in this article demonstrate that Ang-(1-7 increases in plasma and urine of normal pregnant women. In preeclamptic subjects we showed that plasma Ang-(1-7 was suppressed as compared to the levels found in normal pregnancy. In addition, kidney and urinary levels of Ang-(1-7 were increased in pregnant rats coinciding with the enhanced detection and expression of ACE2. These findings support the concept that in normal pregnancy enhanced ACE2 may counteract the elevation in tissue and circulating Ang II by increasing the rate of conversion to Ang-(1-7. These findings provide a basis for the physiological role of Ang-(1-7 and ACE2 during pregnancy.

Ultrasonic destruction of albumin microbubbles enhances gene transfection and expression in cardiac myocytes.

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Wang, Guo-zhong; Liu, Jing-hua; Lü, Shu-zheng; Lü, Yun; Guo, Cheng-jun; Zhao, Dong-hui; Fang, Dong-ping; He, Dong-fang; Zhou, Yuan; Ge, Chang-jiang

2011-05-01

It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes. The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed. The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced β-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05). Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into

GM-CSF enhances tumor invasion by elevated MMP-2, -9, and -26 expression

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Gutschalk, Claudia M; Yanamandra, Archana K; Linde, Nina; Meides, Alice; Depner, Sofia; Mueller, Margareta M

2013-01-01

Granulocyte–macrophage colony-stimulating factor (GM-CSF) promotes tumor progression in different tumor models in an autocrine and paracrine manner. However, at the same time GM-CSF is used in cancer therapies to ameliorate neutropenia. We have previously shown in GM-CSF and G-CSF expressing or negative skin or head and neck squamous cell carcinoma that GM-CSF expression is associated with a highly angiogenic and invasive tumor phenotype. To determine the functional contribution of GM-CSF to tumor invasion, we stably transfected a GM-CSF negative colon adenocarcinoma cell line HT-29 with GM-CSF or treated the same cell line with exogenous GM-CSF. While GM-CSF overexpression and treatment reduced tumor cell proliferation and tumor growth in vitro and in vivo, respectively, it contributed to tumor progression. Together with an enhanced migratory capacity in vitro, we observed a striking increase in tumor cell invasion into the surrounding tissue concomitant with the induction of an activated tumor stroma in GM-CSF overexpressing or GM-CSF treated tumors. In a complex 3D in vitro model, enhanced GM-CSF expression was associated with a discontinued basement membrane deposition that might be mediated by the increased expression and activation of MMP-2, -9, and -26. Treatment with GM-CSF blocking antibodies reversed this effect. The increased presence and activity of these tumor cell derived proteases was confirmed in vivo. Here, expression of MMP-26 protein was predominantly located in pre- and early-invasive areas suggesting MMP-26 expression as an early event in promoting GM-CSF dependent tumor invasion

Facial Expression Enhances Emotion Perception Compared to Vocal Prosody: Behavioral and fMRI Studies.

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Zhang, Heming; Chen, Xuhai; Chen, Shengdong; Li, Yansong; Chen, Changming; Long, Quanshan; Yuan, Jiajin

2018-05-09

Facial and vocal expressions are essential modalities mediating the perception of emotion and social communication. Nonetheless, currently little is known about how emotion perception and its neural substrates differ across facial expression and vocal prosody. To clarify this issue, functional MRI scans were acquired in Study 1, in which participants were asked to discriminate the valence of emotional expression (angry, happy or neutral) from facial, vocal, or bimodal stimuli. In Study 2, we used an affective priming task (unimodal materials as primers and bimodal materials as target) and participants were asked to rate the intensity, valence, and arousal of the targets. Study 1 showed higher accuracy and shorter response latencies in the facial than in the vocal modality for a happy expression. Whole-brain analysis showed enhanced activation during facial compared to vocal emotions in the inferior temporal-occipital regions. Region of interest analysis showed a higher percentage signal change for facial than for vocal anger in the superior temporal sulcus. Study 2 showed that facial relative to vocal priming of anger had a greater influence on perceived emotion for bimodal targets, irrespective of the target valence. These findings suggest that facial expression is associated with enhanced emotion perception compared to equivalent vocal prosodies.

Enhanced human papillomavirus type 8 oncogene expression levels are crucial for skin tumorigenesis in transgenic mice

International Nuclear Information System (INIS)

Hufbauer, M.; Lazic, D.; Akguel, B.; Brandsma, J.L.; Pfister, H.; Weissenborn, S.J.

2010-01-01

Human papillomavirus 8 (HPV8) is involved in skin cancer development in epidermodysplasia verruciformis patients. Transgenic mice expressing HPV8 early genes (HPV8-CER) developed papillomas, dysplasias and squamous cell carcinomas. UVA/B-irradiation and mechanical wounding of HPV8-CER mouse skin led to prompt papilloma induction in about 3 weeks. The aim of this study was to analyze the kinetics and level of transgene expression in response to skin irritations. Transgene expression was already enhanced 1 to 2 days after UVA/B-irradiation or tape-stripping and maintained during papilloma development. The enhanced transgene expression could be assigned to UVB and not to UVA. Papilloma development was thus always paralleled by an increased transgene expression irrespective of the type of skin irritation. A knock-down of E6 mRNA by tattooing HPV8-E6-specific siRNA led to a delay and a lower incidence of papilloma development. This indicates that the early increase of viral oncogene expression is crucial for induction of papillomatosis.

Pimecrolimus enhances TLR2/6-induced expression of antimicrobial peptides in keratinocytes.

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Büchau, Amanda S; Schauber, Jürgen; Hultsch, Thomas; Stuetz, Anton; Gallo, Richard L

2008-11-01

Calcineurin inhibitors are potent inhibitors of T-cell-receptor mediated activation of the adaptive immune system. The effects of this class of drug on the innate immune response system are not known. Keratinocytes are essential to innate immunity in skin and rely on toll-like receptors (TLRs) and antimicrobial peptides to appropriately recognize and respond to injury or microbes. In this study we examined the response of cultured human keratinocytes to pimecrolimus. We observed that pimecrolimus enhances distinct expression of cathelicidin, CD14, and human beta-defensin-2 and beta-defensin-3 in response to TLR2/6 ligands. Some of these responses were further enhanced by 1,25 vitamin D3. Pimecrolimus also increased the functional capacity of keratinocytes to inhibit growth of Staphylococcus aureus and decreased TLR2/6-induced expression of IL-10 and IL-1beta. Furthermore, pimecrolimus inhibited nuclear translocation of NFAT and NF-kappaB in keratinocytes. These observations uncover a previously unreported function for pimecrolimus in cutaneous innate host defense.

Promotion of seminomatous tumors by targeted overexpression of glial cell line-derived neurotrophic factor in mouse testis

NARCIS (Netherlands)

Meng, X.; de rooij, D. G.; Westerdahl, K.; Saarma, M.; Sariola, H.

2001-01-01

We show with transgenic mice that targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) in undifferentiated spermatogonia promotes malignant testicular tumors, which express germ-cell markers. The tumors are invasive and contain aneuploid cells, but no distant metastases have

Comparison of GFL–GFRα complexes: further evidence relating GFL bend angle to RET signalling

International Nuclear Information System (INIS)

Parkash, Vimal; Goldman, Adrian

2009-01-01

The second crystal structure of the GDNF-GFRα1 complex provides further evidence that GFL signalling through RET is determined by the bend angle in the GFL. Glial cell line-derived neurotrophic factor (GDNF) activates the receptor tyrosine kinase RET by binding to the GDNF-family receptor α1 (GFRα1) and forming the GDNF 2 –GFRα1 2 –RET 2 heterohexamer complex. A previous crystal structure of the GDNF 2 –GFRα1 2 complex suggested that differences in signalling in GDNF-family ligand (GFL) complexes might arise from differences in the bend angle between the two monomers in the GFL homodimer. Here, a 2.35 Å resolution structure of the GDNF 2 –GFRα1 2 complex crystallized with new cell dimensions is reported. The structure was refined to a final R factor of 22.5% (R free = 28%). The structures of both biological tetrameric complexes in the asymmetric unit are very similar to 2v5e and different from the artemin–GFRα3 structure, even though there is a small change in the structure of the GDNF. By comparison of all known GDNF and artemin structures, it is concluded that GDNF is more bent and more flexible than artemin and that this may be related to RET signalling. Comparisons also suggest that the differences between artemin and GDNF arise from the increased curvature of the artemin ‘fingers’, which both increases the buried surface area in the monomer–monomer interface and changes the intermonomer bend angle. From sequence comparison, it is suggested that neuturin (the second GFL) adopts an artemin-like conformation, while persephin has a different conformation to the other three

Expression of an isoflavone reductase-like gene enhanced by pollen tube growth in pistils of Solanum tuberosum.

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van Eldik, G J; Ruiter, R K; Colla, P H; van Herpen, M M; Schrauwen, J A; Wullems, G J

1997-03-01

Successful sexual reproduction relies on gene products delivered by the pistil to create an environment suitable for pollen tube growth. These compounds are either produced before pollination or formed during the interactions between pistil and pollen tubes. Here we describe the pollination-enhanced expression of the cp100 gene in pistils of Solanum tuberosum. Temporal analysis of gene expression revealed an enhanced expression already one hour after pollination and lasts more than 72 h. Increase in expression also occurred after touching the stigma and was not restricted to the site of touch but spread into the style. The predicted CP100 protein shows similarity to leguminous isoflavone reductases (IFRs), but belongs to a family of IFR-like NAD(P)H-dependent oxidoreductases present in various plant species.

Enhanced expressions of microvascular smooth muscle receptors after focal cerebral ischemia occur via the MAPK MEK/ERK pathway

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Edvinsson Lars

2008-09-01

Full Text Available Abstract Background MEK1/2 is a serine/threonine protein that phosphorylates extracellular signal-regulated kinase (ERK1/2. Cerebral ischemia results in enhanced expression of cerebrovascular contractile receptors in the middle cerebral artery (MCA leading to the ischemic region. Here we explored the role of the MEK/ERK pathway in receptor expression following ischemic brain injury using the specific MEK1 inhibitor U0126. Methods and result Rats were subjected to a 2-h middle cerebral artery occlusion (MCAO followed by reperfusion for 48-h and the ischemic area was calculated. The expression of phosphorylated ERK1/2 and Elk-1, and of endothelin ETA and ETB, angiotensin AT1, and 5-hydroxytryptamine 5-HT1B receptors were analyzed with immunohistochemistry using confocal microscopy in cerebral arteries, microvessels and in brain tissue. The expression of endothelin ETB receptor was analyzed by quantitative Western blot. We demonstrate that there is an increase in the number of contractile smooth muscle receptors in the MCA and in micro- vessels within the ischemic region. The enhanced expression occurs in the smooth muscle cells as verified by co-localization studies. This receptor upregulation is furthermore associated with enhanced expression of pERK1/2 and of transcription factor pElk-1 in the vascular smooth muscle cells. Blockade of transcription with the MEK1 inhibitor U0126, given at the onset of reperfusion or as late as 6 hours after the insult, reduced transcription (pERK1/2 and pElk-1, the enhanced vascular receptor expression, and attenuated the cerebral infarct and improved neurology score. Conclusion Our results show that MCAO results in upregulation of cerebrovascular ETB, AT1 and 5-HT1B receptors. Blockade of this event with a MEK1 inhibitor as late as 6 h after the insult reduced the enhanced vascular receptor expression and the associated cerebral infarction.

Enhanced insulin-like growth factor I gene expression in regenerating rat pancreas

International Nuclear Information System (INIS)

Smith, F.E.; Rosen, K.M.; Villa-Komaroff, L.; Weir, G.C.; Bonner-Weir, S.

1991-01-01

Insulin-like growth factor I (IGF-I) mRNA expression was studied after 90% partial pancreatectomy in the rat to determine whether IGF-I was associated with pancreatic regeneration. The level of IGF-I mRNA was maximally increased (4-fold above control value) 3 days after pancreatectomy, but thereafter gradually decreased, returning to control levels by 14 days after surgery. By in situ hybridization, IGF-I mRNA in both pancreatectomized and sham-operated rats was localized to capillary endothelial cells, indicating that this is the site of IGF-I expression in the normal rat pancreas. However, enhanced IGF-I mRNA expression was localized to focal areas of regeneration unique to pancreatectomized rats. In these areas, epithelial cells of proliferating ductules and individual connective tissue cells expressed IGF-I, suggesting that IGF-I may play an important role in the growth or differentiation of pancreatic tissue

Enhanced insulin-like growth factor I gene expression in regenerating rat pancreas

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Smith, F.E.; Rosen, K.M.; Villa-Komaroff, L.; Weir, G.C.; Bonner-Weir, S. (E. P. Joslin Research Laboratory, Joslin Diabetes Center, Harvard Medical School, Boston, MA (USA))

1991-07-15

Insulin-like growth factor I (IGF-I) mRNA expression was studied after 90% partial pancreatectomy in the rat to determine whether IGF-I was associated with pancreatic regeneration. The level of IGF-I mRNA was maximally increased (4-fold above control value) 3 days after pancreatectomy, but thereafter gradually decreased, returning to control levels by 14 days after surgery. By in situ hybridization, IGF-I mRNA in both pancreatectomized and sham-operated rats was localized to capillary endothelial cells, indicating that this is the site of IGF-I expression in the normal rat pancreas. However, enhanced IGF-I mRNA expression was localized to focal areas of regeneration unique to pancreatectomized rats. In these areas, epithelial cells of proliferating ductules and individual connective tissue cells expressed IGF-I, suggesting that IGF-I may play an important role in the growth or differentiation of pancreatic tissue.

Can expressions of anger enhance creativity? A test of the emotions as social information (EASI) model

NARCIS (Netherlands)

van Kleef, Gerben A.; Anastasopoulou, Christina; Nijstad, Bernard A.

We investigated whether expressions of anger can enhance creative performance. Building on the emotions as social information (EASI) model (Van Kleef, 2009), we predicted that the interpersonal effects of anger expressions on creativity depend on the target's epistemic motivation (EM) the desire to

A Novel Growth-Promoting Pathway Formed by GDNF-Overexpressing Schwann Cells Promotes Propriospinal Axonal Regeneration, Synapse formation, and Partial Recovery of Function after Spinal Cord Injury

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Deng, Lingxiao; Deng, Ping; Ruan, Yiwen; Xu, Zao Cheng; Liu, Naikui; Wen, Xuejun; Smith, George M.; Xu, Xiao-Ming

2013-01-01

Descending propriospinal neurons (DPSN) are known to establish functional relays for supraspinal signals, and they display a greater growth response after injury than do the long projecting axons. However, their regenerative response is still deficient due to their failure to depart from growth supportive cellular transplants back into the host spinal cord, which contains numerous impediments to axon growth. Here we report the construction of a continuous growth-promoting pathway in adult rats, formed by grafted Schwann cells (SCs) overexpressing glial cell line-derived neurotrophic factor (GDNF). We demonstrate that such a growth-promoting pathway, extending from the axonal cut ends to the site of innervation in the distal spinal cord, promoted regeneration of DPSN axons through and beyond the lesion gap of a spinal cord hemisection. Within the distal host spinal cord, regenerated DPSN axons formed synapses with host neurons leading to the restoration of action potentials and partial recovery of function. PMID:23536080

Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death

International Nuclear Information System (INIS)

Porter, Holly A.; Carey, Gregory B.; Keegan, Achsah D.

2012-01-01

The adapters IRS1 and IRS2 link growth factor receptors to downstream signaling pathways that regulate proliferation and survival. Both suppress factor-withdrawal-induced apoptosis and have been implicated in cancer progression. However, recent studies suggest IRS1 and IRS2 mediate differential functions in cancer pathogenesis. IRS1 promoted breast cancer proliferation, while IRS2 promoted metastasis. The role of IRS1 and IRS2 in controlling cell responses to chemotherapy is unknown. To determine the role of IRS1 and IRS2 in the sensitivity of cells to chemotherapy, we treated 32D cells lacking or expressing IRS proteins with various concentrations of chemotherapeutic agents. We found that expression of IRS1, in contrast to IRS2, enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. When IRS2 was expressed with IRS1, the cells no longer showed enhanced sensitivity. Expression of IRS1 did not alter the expression of pro- and anti-apoptotic proteins; however, 32D-IRS1 cells expressed higher levels of Annexin A2. In 32D-IRS1 cells, IRS1 and Annexin A2 were both located in cytoplasmic and membrane fractions. We also found that IRS1 coprecipitated with Annexin A2, while IRS2 did not. Decreasing Annexin A2 levels reduced 32D-IRS1 cell sensitivity to chemotherapy. These results suggest IRS1 enhances sensitivity to chemotherapy in part through Annexin A2. -- Highlights: ► IRS1 enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. ► This sensitivity is abrogated by the expression of IRS2. ► Expressing IRS1 in 32D cells increased levels of Annexin A2. ► Both IRS1 and Annexin A2 were located in cytoplasmic and membrane fractions. ► Decreasing Annexin A2 in 32D-IRS1 cells abated their sensitivity to chemotherapy.

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius

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Christopher D Johnston

2014-09-01

Full Text Available It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of two MAP genes (MAP2121c and MAP3733c can enhance the heterologous expression of two antigens (MMP and MptD respectively, analogous to the form to which they are produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, codon optimised MptD displayed the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adhered with the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne’s disease.

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius.

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Johnston, Christopher D; Bannantine, John P; Govender, Rodney; Endersen, Lorraine; Pletzer, Daniel; Weingart, Helge; Coffey, Aidan; O'Mahony, Jim; Sleator, Roy D

2014-01-01

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.

Can expressions of anger enhance creativity? A test of the emotions as social information (EASI) model

NARCIS (Netherlands)

van Kleef, G.A.; Anastasopoulou, C.; Nijstad, B.A.

2010-01-01

We investigated whether expressions of anger can enhance creative performance. Building on the emotions as social information (EASI) model (Van Kleef, 2009), we predicted that the interpersonal effects of anger expressions on creativity depend on the target's epistemic motivation (EM)—the desire to

Enhanced Chemokine Receptor Expression on Leukocytes of Patients with Alzheimer's Disease.

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David Goldeck

Full Text Available Although primarily a neurological complaint, systemic inflammation is present in Alzheimer's Disease, with higher than normal levels of proinflammatory cytokines and chemokines in the periphery as well as the brain. A gradient of these factors may enhance recruitment of activated immune cells into the brain via chemotaxis. Here, we investigated the phenotypes of circulating immune cells in AD patients with multi-colour flow cytometry to determine whether their expression of chemokine receptors is consistent with this hypothesis. In this study, we confirmed our previously reported data on the shift of early- to late-differentiated CD4+ T-cells in AD patients. The percentage of cells expressing CD25, a marker of acute T-cell activation, was higher in patients than in age-matched controls, and percentages of CCR6+ cells were elevated. This chemokine receptor is primarily expressed on pro-inflammatory memory cells and Th17 cells. The proportion of cells expressing CCR4 (expressed on Th2 cells and CCR5 (Th1 cells and dendritic cells was also greater in patients, and was more pronounced on CD4+ than CD8+ T-cells. These findings allow a more detailed insight into the systemic immune status of patients with Alzheimer's disease and suggest possible novel targets for immune therapy.

Stress Hormone Cortisol Enhances Bcl2 Like-12 Expression to Inhibit p53 in Hepatocellular Carcinoma Cells.

Science.gov (United States)

Wu, Weizhong; Liu, Sanguang; Liang, Yunfei; Zhou, Zegao; Bian, Wei; Liu, Xueqing

2017-12-01

The pathogenesis of hepatocellular carcinoma (HC) is unclear. It is suggested that psychological stress associates with the pathogenesis of liver cancer. Bcl2-like protein 12 (Bcl2L12) suppresses p53 protein. This study tests a hypothesis that the major stress hormone, cortisol, inhibits the expression of p53 in HC cells (HCC) via up regulating the expression of Bcl2L12. Peripheral blood samples were collected from patients with HC to be analyzed for the levels of cortisol. HCC were cultured to assess the role of cortisol in the regulation of the expression of Bcl2L12 and p53 in HCC. We observed that the serum cortisol levels were higher in HC patients. Expression of Bcl2L12 in HCC was correlated with serum cortisol. Cortisol enhanced the Bcl2L12 expression in HCC. Bcl2L12 binding to the TP53 promoter was correlated with p53 expression in HCC. Cortisol increased the Bcl2L12 expression in HCC to inhibit p53 expression. Stress hormone cortisol suppresses p53 in HCC via enhancing Bcl2L12 expression in HCC. The results suggest that cortisol may be a therapeutic target for the treatment of HC.

Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study.

Science.gov (United States)

Chlapek, Petr; Redova, Martina; Zitterbart, Karel; Hermanova, Marketa; Sterba, Jaroslav; Veselska, Renata

2010-05-11

We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2) or SH-SY5Y) after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX) in combination with ATRA in both cell lines. Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

Chicken IgY Fc expressed by Eimeria mitis enhances the immunogenicity of E. mitis.

Science.gov (United States)

Qin, Mei; Tang, Xinming; Yin, Guangwen; Liu, Xianyong; Suo, Jingxia; Tao, Geru; Ei-Ashram, Saeed; Li, Yuan; Suo, Xun

2016-03-21

Eimeria species are obligate intracellular apicomplexan parasites, causing great economic losses in the poultry industry. Currently wild-and attenuated- type anticoccidial vaccines are used to control coccidiosis. However, their use in fast growing broilers is limited by vaccination side effects caused by medium and/or low immunogenic Eimeria spp. There is, therefore, a need for a vaccine with high immunogenicity for broilers. The avian yolk sac IgY Fc is the avian counterpart of the mammalian IgG Fc, which enhances immunogenicity of Fc-fusion proteins. Here, we developed a stable transgenic Eimeria mitis expressing IgY Fc (Emi.chFc) and investigated whether the avian IgY Fc fragment enhances the immunogenicity of E. mitis. Two-week-old broilers were immunized with either Emi.chFc or wild type Eimeria and challenged with wild type E. mitis to analyze the protective properties of transgenic Emi.chFc. Chickens immunized with Emi.chFc had significantly lower oocyst output, in comparison with PBS, mock control (transgenic E. mitis expressing HA1 from H9N2 avian influenza virus) and wildtype E. mitis immunized groups after challenge, indicating that IgY Fc enhanced the immunogenicity of E. mitis. Our findings suggest that IgY Fc-expressing Eimeria may be a better coccidiosis vaccine, and transgenic Eimeria expressing Fc-fused exogenous antigens may be used as a novel vaccine-delivery vehicle against a wide variety of pathogens.

Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity.

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Hans Rempel

2008-04-01

Full Text Available HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14(+ monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1, a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases.We analyzed sialoadhesin expression on CD14(+ monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14(+ monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-alpha and interferon-gamma but not tumor necrosis factor-alpha. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection.Increased sialoadhesin expression on CD14(+ monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14(+ monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells.

Inhibition of COX-2 expression by topical diclofenac enhanced radiation sensitivity via enhancement of TRAIL in human prostate adenocarcinoma xenograft model

Science.gov (United States)

2013-01-01

Background COX-2 inhibitors have an antitumor potential and have been verified by many researchers. Treatment of cancer cells with external stressors such as irradiation can stimulate the over-expression of COX-2 and possibly confer radiation resistance. In this study, we tested if topical diclofenac, which inhibits both COX-1 and COX-2, administration rendered prostate tumor cells sensitize to the effects of radiation. Methods LNCaP-COX-2 and LNCaP-Neo cells were treated with 0 to 1000 μM diclofenac. Next, a clonogenic assay was performed in which cells were subjected to irradiation (0 to 4 Gy) with or without diclofenac. COX-2 expression and other relevant molecules were measured by real-time PCR and immunohistochemistry after irradiation and diclofenac treatment. In addition, we assessed the tumor volumes of xenograft LNCaP-COX-2 cells treated with topical diclofenac with or without radiation therapy (RT). Results LNCaP-COX-2 and LNCaP-Neo cell lines experienced cytotoxic effects of diclofenac in a dose related manner. Clonogenic assays demonstrated that LNCaP-COX-2 cells were significantly more resistant to RT than LNCaP-Neo cells. Furthermore, the addition of diclofenac sensitized LNCaP-COX-2 not but LNCaP-Neo cells to the cytocidal effects of radiation. In LNCaP-COX-2 cells, diclofenac enhanced radiation-induced apoptosis compared with RT alone. This phenomenon might be attributed to enhancement of RT-induced TRAIL expression as demonstrated by real-time PCR analysis. Lastly, tumor volumes of LNCaP-COX-2 cells xenograft treated with diclofenac or RT alone was >4-fold higher than in mice treated with combined diclofenac and radiation (pdiclofenac enhances the effect of RT on prostate cancer cells that express COX-2. Thus, diclofenac may have potential as radiosensitizer for treatment of prostate cancer. PMID:23289871

Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death

Science.gov (United States)

Porter, Holly A.; Carey, Gregory B.; Keegan, Achsah D.

2012-01-01

The adaptors IRS1 and IRS2 link growth factor receptors to downstream signaling pathways that regulate proliferation and survival. Both suppress factor-withdrawal-induced apoptosis and have been implicated in cancer progression. However, recent studies suggest IRS1 and IRS2 mediate differential functions in cancer pathogenesis. IRS1 promoted breast cancer proliferation, while IRS2 promoted metastasis. The role of IRS1 and IRS2 in controlling cell responses to chemotherapy is unknown. To determine the role of IRS1 and IRS2 in the sensitivity of cells to chemotherapy, we treated 32D cells lacking or expressing IRS proteins with various concentrations of chemotherapeutic agents. We found that expression of IRS1, in contrast to IRS2, enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. When IRS2 was expressed with IRS1, the cells no longer showed enhanced sensitivity. Expression of IRS1 did not alter the expression of pro- and anti-apoptotic proteins; however, 32D-IRS1 cells expressed higher levels of Annexin A2. In 32D-IRS1 cells, IRS1 and Annexin A2 were both located in cytoplasmic and membrane fractions. We also found that IRS1 coprecipitated with Annexin A2, while IRS2 did not. Decreasing Annexin A2 levels reduced 32D-IRS1 cell sensitivity to chemotherapy. These results suggest IRS1 enhances sensitivity to chemotherapy in part through Annexin A2. PMID:22652453

Ethanol enhances arsenic-induced cyclooxygenase-2 expression via both NFAT and NF-κB signalings in colorectal cancer cells

Energy Technology Data Exchange (ETDEWEB)

Wang, Lei; Hitron, John Andrew [Center for Research on Environmental Disease, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Wise, James T.F. [Pharmacology and Nutritional Sciences, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Son, Young-Ok; Roy, Ram Vinod [Center for Research on Environmental Disease, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Kim, Donghern; Dai, Jin [Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Pratheeshkumar, Poyil [Center for Research on Environmental Disease, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Zhang, Zhuo [Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Xu, Mei; Luo, Jia [Pharmacology and Nutritional Sciences, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Shi, Xianglin, E-mail: xshi5@uky.edu [Center for Research on Environmental Disease, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States)

2015-10-15

Arsenic is a known carcinogen to humans, and chronic exposure to environmental arsenic is a worldwide health concern. As a dietary factor, ethanol carries a well-established risk for malignancies, but the effects of co-exposure to arsenic and ethanol on tumor development are not well understood. In the present study, we hypothesized that ethanol would enhance the function of an environmental carcinogen such as arsenic through increase in COX-2 expression. Our in vitro results show that ethanol enhanced arsenic-induced COX-2 expression. We also show that the increased COX-2 expression associates with intracellular ROS generation, up-regulated AKT signaling, with activation of both NFAT and NF-κB pathways. We demonstrate that antioxidant enzymes have an inhibitory effect on arsenic/ethanol-induced COX-2 expression, indicating that the responsive signaling pathways from co-exposure to arsenic and ethanol relate to ROS generation. In vivo results also show that co-exposure to arsenic and ethanol increased COX-2 expression in mice. We conclude that ethanol enhances arsenic-induced COX-2 expression in colorectal cancer cells via both the NFAT and NF-κB pathways. These results imply that, as a common dietary factor, ethanol ingestion may be a compounding risk factor for arsenic-induced carcinogenesis/cancer development. - Highlights: • Arsenic is able to induce Cox-2 expression in colorectal cancer cells. • Ethanol, a diet nutritional factor, could enhance arsenic-induced Cox-2. • The up-regulation of Cox-2 via both NFAT and NF-κB activities.

Ethanol enhances arsenic-induced cyclooxygenase-2 expression via both NFAT and NF-κB signalings in colorectal cancer cells

International Nuclear Information System (INIS)

Wang, Lei; Hitron, John Andrew; Wise, James T.F.; Son, Young-Ok; Roy, Ram Vinod; Kim, Donghern; Dai, Jin; Pratheeshkumar, Poyil; Zhang, Zhuo; Xu, Mei; Luo, Jia; Shi, Xianglin

2015-01-01

Arsenic is a known carcinogen to humans, and chronic exposure to environmental arsenic is a worldwide health concern. As a dietary factor, ethanol carries a well-established risk for malignancies, but the effects of co-exposure to arsenic and ethanol on tumor development are not well understood. In the present study, we hypothesized that ethanol would enhance the function of an environmental carcinogen such as arsenic through increase in COX-2 expression. Our in vitro results show that ethanol enhanced arsenic-induced COX-2 expression. We also show that the increased COX-2 expression associates with intracellular ROS generation, up-regulated AKT signaling, with activation of both NFAT and NF-κB pathways. We demonstrate that antioxidant enzymes have an inhibitory effect on arsenic/ethanol-induced COX-2 expression, indicating that the responsive signaling pathways from co-exposure to arsenic and ethanol relate to ROS generation. In vivo results also show that co-exposure to arsenic and ethanol increased COX-2 expression in mice. We conclude that ethanol enhances arsenic-induced COX-2 expression in colorectal cancer cells via both the NFAT and NF-κB pathways. These results imply that, as a common dietary factor, ethanol ingestion may be a compounding risk factor for arsenic-induced carcinogenesis/cancer development. - Highlights: • Arsenic is able to induce Cox-2 expression in colorectal cancer cells. • Ethanol, a diet nutritional factor, could enhance arsenic-induced Cox-2. • The up-regulation of Cox-2 via both NFAT and NF-κB activities.

Estrogen enhances mismatch repair by induction of MLH1 expression via estrogen receptor-β.

Science.gov (United States)

Lu, Jun-Yu; Jin, Peng; Gao, Wei; Wang, De-Zhi; Sheng, Jian-Qiu

2017-06-13

Epidemiological data demonstrated that hormone replace treatment has protective effect against colorectal cancer (CRC). Our previous studies showed that this effect may be associated with DNA mismatch repair. This study aims to investigate the mechanism of estrogen induction of MLH1, and whether colorectal tumor proliferation can be inhibited through induction of MLH1 by estrogen signal pathway. Human CRC cell lines were used to examine the regulation of MLH1 expression by over-expression and depletion of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ), under the treatment with 17β-estradiol or β-Estradiol 6-(O-carboxy-methyl)oxime:BSA, followed by a real-time Q-PCR and Western blotting analysis. Luciferase reporter and chromatin immunoprecipitation assays were used to identify the estrogen response elements in the proximal promoter of MLH1 gene. Then, the influence of estrogen-induced MLH1 on CRC tumor growth were determined in vitro and in vivo. We found that mismatch repair ability and microsatellite stability of cells were enhanced by estrogen via induction of MLH1 expression, which was mediated by ERβ, through a transcriptional activation process. Furthermore, we identified that ERβ exerted an inhibitory effect on CRC tumor proliferation in vitro and in vivo, combined with 5-FU, through up-regulation of MLH1 expression. Finally, we concluded that estrogen enhances mismatch repair ability and tumor inhibition effect in vitro and in vivo, via induction of MLH1 expression mediated by ERβ.

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells.

Science.gov (United States)

Alvarez-Guerra, Miriam; Hernandez, Maria Rosa; Escolar, Ginés; Chiavaroli, Carlo; Garay, Ricardo P; Hannaert, Patrick

2002-09-15

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance. Copyright 2002 Elsevier Science Ltd.

Induction of neurite outgrowth in 3D hydrogel-based environments

International Nuclear Information System (INIS)

Assunção-Silva, Rita C; Oliveira, Cátia Costa; Gomes, Eduardo D; Sousa, Nuno; Silva, Nuno A; Salgado, António J; Ziv-Polat, Ofra; Sahar, Abraham

2015-01-01

The ability of peripheral nervous system (PNS) axons to regenerate and re-innervate their targets after an injury has been widely recognized. However, despite the considerable advances made in microsurgical techniques, complete functional recovery is rarely achieved, especially for severe peripheral nerve injuries (PNIs). Therefore, alternative therapies that can successfully repair peripheral nerves are still essential. In recent years the use of biodegradable hydrogels enriched with growth-supporting and guidance cues, cell transplantation, and biomolecular therapies have been explored for the treatment of PNIs. Bearing this in mind, the aim of this study was to assess whether Gly-Arg-Gly-Asp-Ser synthetic peptide (GRGDS)-modified gellan gum (GG) based hydrogels could foster an amenable environment for neurite/axonal growth. Additionally, strategies to further improve the rate of neurite outgrowth were also tested, namely the use of adipose tissue derived stem cells (ASCs), as well as the glial derived neurotrophic factor (GDNF). In order to increase its stability and enhance its bioactivity, the GDNF was conjugated covalently to iron oxide nanoparticles (IONPs). The impact of hydrogel modification as well as the effect of the GDNF-IONPs on ASC behavior was also screened. The results revealed that the GRGDS-GG hydrogel was able to support dorsal root ganglia (DRG)-based neurite outgrowth, which was not observed for non-modified hydrogels. Moreover, the modified hydrogels were also able to support ASCs attachment. In contrast, the presence of the GDNF-IONPs had no positive or negative impact on ASC behavior. Further experiments revealed that the presence of ASCs in the hydrogel improved axonal growth. On the other hand, GDNF-IONPs alone or combined with ASCs significantly increased neurite outgrowth from DRGs, suggesting a beneficial role of the proposed strategy for future applications in PNI regenerative medicine. (note)

A mammalian conserved element derived from SINE displays enhancer properties recapitulating Satb2 expression in early-born callosal projection neurons.

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Kensuke Tashiro

Full Text Available Short interspersed repetitive elements (SINEs are highly repeated sequences that account for a significant proportion of many eukaryotic genomes and are usually considered "junk DNA". However, we previously discovered that many AmnSINE1 loci are evolutionarily conserved across mammalian genomes, suggesting that they may have acquired significant functions involved in controlling mammalian-specific traits. Notably, we identified the AS021 SINE locus, located 390 kbp upstream of Satb2. Using transgenic mice, we showed that this SINE displays specific enhancer activity in the developing cerebral cortex. The transcription factor Satb2 is expressed by cortical neurons extending axons through the corpus callosum and is a determinant of callosal versus subcortical projection. Mouse mutants reveal a crucial function for Sabt2 in corpus callosum formation. In this study, we compared the enhancer activity of the AS021 locus with Satb2 expression during telencephalic development in the mouse. First, we showed that the AS021 enhancer is specifically activated in early-born Satb2(+ neurons. Second, we demonstrated that the activity of the AS021 enhancer recapitulates the expression of Satb2 at later embryonic and postnatal stages in deep-layer but not superficial-layer neurons, suggesting the possibility that the expression of Satb2 in these two subpopulations of cortical neurons is under genetically distinct transcriptional control. Third, we showed that the AS021 enhancer is activated in neurons projecting through the corpus callosum, as described for Satb2(+ neurons. Notably, AS021 drives specific expression in axons crossing through the ventral (TAG1(-/NPY(+ portion of the corpus callosum, confirming that it is active in a subpopulation of callosal neurons. These data suggest that exaptation of the AS021 SINE locus might be involved in enhancement of Satb2 expression, leading to the establishment of interhemispheric communication via the corpus callosum

Titania nanotube delivery fetal bovine serum for enhancing MC3T3-E1 activity and osteogenic gene expression

International Nuclear Information System (INIS)

Peng, Jing; Zhang, Xinming; Li, Zhaoyang; Liu, Yunde; Yang, Xianjin

2015-01-01

Titania nanotube (TNT) delivery of fetal bovine serum (FBS) was conducted on titanium (Ti) to enhance bone tissue repair. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) showed FBS increased the tube wall thickness and decreased the tube diameter. Attenuated total reflectance Fourier transform infrared further confirmed that FBS completely covered the TNT and changed the surface composition. Water contact angle tests showed TNT/FBS possessed hydrophilic properties. Compared to original Ti, the TNT/FBS group had more attached osteoblasts after 2 h and enhanced filopodia growth at 0.5 h. Significantly, more osteoblasts were also observed on TNT/FBS after 7 d culturing. FBS was released steadily from TNT; about 70% of FBS had been released at 3 d and 90% at 5 d, as shown by the bicinchoninic acid method. TNT/FBS also enhanced subsequent osteoblast differentiation and gene expression; the quantum real-time polymerase chain reaction test showed that TNT/FBS up-regulated alkaline phosphatase and osteocalcin gene expression at 7 d and 14 d. Therefore, TNT/FBS delivered sustained in situ nutrition and enhanced osteoblast activity and osteogenic gene expression. - Highlights: • Fetal Bovine Serum (FBS) was filled in titania nanotube (TNT) structures. • FBS provided sustained-release in situ nutrition for surface osteoblast growth. • TNT/FBS enhanced osteoblast activity and osteogenic gene expression

Titania nanotube delivery fetal bovine serum for enhancing MC3T3-E1 activity and osteogenic gene expression

Energy Technology Data Exchange (ETDEWEB)

Peng, Jing, E-mail: pengjingtd@163.com [Airport College, Civil Aviation University of China, Tianjin 300300 (China); Zhang, Xinming, E-mail: xinmingmail@163.com [Tianjin Product Quality Inspection Technology Research Institute, Tianjin 300384 (China); School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Li, Zhaoyang [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Liu, Yunde [School of Medical Laboratory, Tianjin Medical University, Tianjin 300203 (China); Yang, Xianjin [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China)

2015-11-01

Titania nanotube (TNT) delivery of fetal bovine serum (FBS) was conducted on titanium (Ti) to enhance bone tissue repair. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) showed FBS increased the tube wall thickness and decreased the tube diameter. Attenuated total reflectance Fourier transform infrared further confirmed that FBS completely covered the TNT and changed the surface composition. Water contact angle tests showed TNT/FBS possessed hydrophilic properties. Compared to original Ti, the TNT/FBS group had more attached osteoblasts after 2 h and enhanced filopodia growth at 0.5 h. Significantly, more osteoblasts were also observed on TNT/FBS after 7 d culturing. FBS was released steadily from TNT; about 70% of FBS had been released at 3 d and 90% at 5 d, as shown by the bicinchoninic acid method. TNT/FBS also enhanced subsequent osteoblast differentiation and gene expression; the quantum real-time polymerase chain reaction test showed that TNT/FBS up-regulated alkaline phosphatase and osteocalcin gene expression at 7 d and 14 d. Therefore, TNT/FBS delivered sustained in situ nutrition and enhanced osteoblast activity and osteogenic gene expression. - Highlights: • Fetal Bovine Serum (FBS) was filled in titania nanotube (TNT) structures. • FBS provided sustained-release in situ nutrition for surface osteoblast growth. • TNT/FBS enhanced osteoblast activity and osteogenic gene expression.

Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study

Directory of Open Access Journals (Sweden)

Hermanova Marketa

2010-05-01

Full Text Available Abstract Background We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2 and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA and inhibitors of lipoxygenases (LOX and cyclooxygenases (COX. This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Methods Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Results Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2 or SH-SY5Y after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX in combination with ATRA in both cell lines. Conclusions Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression.

Science.gov (United States)

Diani, Erica; Avesani, Francesca; Bergamo, Elisa; Cremonese, Giorgia; Bertazzoni, Umberto; Romanelli, Maria Grazia

2015-02-01

The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression. Copyright © 2014 Elsevier Inc. All rights reserved.

Transgenic Citrus Expressing an Arabidopsis NPR1 Gene Exhibit Enhanced Resistance against Huanglongbing (HLB; Citrus Greening).

Science.gov (United States)

Dutt, Manjul; Barthe, Gary; Irey, Michael; Grosser, Jude

2015-01-01

Commercial sweet orange cultivars lack resistance to Huanglongbing (HLB), a serious phloem limited bacterial disease that is usually fatal. In order to develop sustained disease resistance to HLB, transgenic sweet orange cultivars 'Hamlin' and 'Valencia' expressing an Arabidopsis thaliana NPR1 gene under the control of a constitutive CaMV 35S promoter or a phloem specific Arabidopsis SUC2 (AtSUC2) promoter were produced. Overexpression of AtNPR1 resulted in trees with normal phenotypes that exhibited enhanced resistance to HLB. Phloem specific expression of NPR1 was equally effective for enhancing disease resistance. Transgenic trees exhibited reduced diseased severity and a few lines remained disease-free even after 36 months of planting in a high-disease pressure field site. Expression of the NPR1 gene induced expression of several native genes involved in the plant defense signaling pathways. The AtNPR1 gene being plant derived can serve as a component for the development of an all plant T-DNA derived consumer friendly GM tree.

Transgenic Citrus Expressing an Arabidopsis NPR1 Gene Exhibit Enhanced Resistance against Huanglongbing (HLB; Citrus Greening.

Directory of Open Access Journals (Sweden)

Manjul Dutt

Full Text Available Commercial sweet orange cultivars lack resistance to Huanglongbing (HLB, a serious phloem limited bacterial disease that is usually fatal. In order to develop sustained disease resistance to HLB, transgenic sweet orange cultivars 'Hamlin' and 'Valencia' expressing an Arabidopsis thaliana NPR1 gene under the control of a constitutive CaMV 35S promoter or a phloem specific Arabidopsis SUC2 (AtSUC2 promoter were produced. Overexpression of AtNPR1 resulted in trees with normal phenotypes that exhibited enhanced resistance to HLB. Phloem specific expression of NPR1 was equally effective for enhancing disease resistance. Transgenic trees exhibited reduced diseased severity and a few lines remained disease-free even after 36 months of planting in a high-disease pressure field site. Expression of the NPR1 gene induced expression of several native genes involved in the plant defense signaling pathways. The AtNPR1 gene being plant derived can serve as a component for the development of an all plant T-DNA derived consumer friendly GM tree.

Mutual repression enhances the steepness and precision of gene expression boundaries.

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Thomas R Sokolowski

Full Text Available Embryonic development is driven by spatial patterns of gene expression that determine the fate of each cell in the embryo. While gene expression is often highly erratic, embryonic development is usually exceedingly precise. In particular, gene expression boundaries are robust not only against intra-embryonic fluctuations such as noise in gene expression and protein diffusion, but also against embryo-to-embryo variations in the morphogen gradients, which provide positional information to the differentiating cells. How development is robust against intra- and inter-embryonic variations is not understood. A common motif in the gene regulation networks that control embryonic development is mutual repression between pairs of genes. To assess the role of mutual repression in the robust formation of gene expression patterns, we have performed large-scale stochastic simulations of a minimal model of two mutually repressing gap genes in Drosophila, hunchback (hb and knirps (kni. Our model includes not only mutual repression between hb and kni, but also the stochastic and cooperative activation of hb by the anterior morphogen Bicoid (Bcd and of kni by the posterior morphogen Caudal (Cad, as well as the diffusion of Hb and Kni between neighboring nuclei. Our analysis reveals that mutual repression can markedly increase the steepness and precision of the gap gene expression boundaries. In contrast to other mechanisms such as spatial averaging and cooperative gene activation, mutual repression thus allows for gene-expression boundaries that are both steep and precise. Moreover, mutual repression dramatically enhances their robustness against embryo-to-embryo variations in the morphogen levels. Finally, our simulations reveal that diffusion of the gap proteins plays a critical role not only in reducing the width of the gap gene expression boundaries via the mechanism of spatial averaging, but also in repairing patterning errors that could arise because of the

Effect of neurturin on multipotent cells isolated from the adult skeletal muscle

International Nuclear Information System (INIS)

Vourc'h, Patrick; Lacar, Benjamin; Mignon, Laurence; Lucas, Paul A.; Young, Henry E.; Chesselet, Marie-Francoise

2005-01-01

Ligands of the glial cell line-derived neurotrophic factors (GDNF)-family are trophic factors for the development and survival of multiple cell types, however their effects on non-neuronal stem cells are unknown. We examined the action of neurturin on a candidate stem cell population isolated from adult skeletal muscles. When grown as spheres, these cells expressed mRNAs for GDNF, persephin, GFR-α2, GFR-α4 (neurturin receptor), and Ret. Exposure of these cells to neurturin significantly augmented cell numbers via increased cell proliferation. After addition of retinoic acid, the cells exited the cell cycle, developed thin processes, and became immunoreactive for βIII-tubulin, while Ret mRNA expression decreased, without changes in the level of GFR-α2 mRNA. Neurturin induced an outgrowth of processes on these βIII-tubulin positive cells. Neurturin may therefore be beneficial in the use of these multipotent cells isolated from adult muscles for autologous transplants in neurological applications

Re-expression of ARHI (DIRAS3) induces autophagy in breast cancer cells and enhances the inhibitory effect of paclitaxel

International Nuclear Information System (INIS)

Zou, Chun-Fang; Yu, Yinhua; Jia, Luoqi; Jin, Hongyan; Yao, Ming; Zhao, Naiqing; Huan, Jin; Lu, Zhen; Bast, Robert C Jr; Feng, Youji

2011-01-01

ARHI is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma in situ (DCIS) to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel. Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA) or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC) in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts. ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest. ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest

Mutual enhancement of IL-2 and IL-7 on DNA vaccine immunogenicity mainly involves regulations on their receptor expression and receptor-expressing lymphocyte generation.

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Zhang, Yonghong; Liang, Shuang; Li, Xiujin; Wang, Liyue; Zhang, Jianlou; Xu, Jian; Huo, Shanshan; Cao, Xuebin; Zhong, Zhenyu; Zhong, Fei

2015-07-09

Our previous study showed that IL-2 and IL-7 could mutually enhance the immunogenicity of canine parvovirus VP2 DNA vaccine, although the underlying mechanism remained unknown. Here, we used the OVA gene as a DNA vaccine in a mouse model to test their enhancement on DNA vaccine immunogenicity and to explore the molecular mechanism. Results showed that both IL-2 and IL-7 genes significantly increased the immunogenicity of OVA DNA vaccine in mice. Co-administration of IL-2 and IL-7 genes with OVA DNA significantly increased OVA-specific antibody titers, T cell proliferation and IFN-γ production compared with IL-2 or IL-7 alone, confirming that IL-2 and IL-7 mutually enhanced DNA vaccine immunogenicity. Mechanistically, we have shown that IL-2 significantly stimulated generation of IL-7 receptor-expressing lymphocytes, and that IL-7 significantly induced IL-2 receptor expression. These results contribute to an explanation of the mechanism of the mutual effects of IL-2 and IL-7 on enhancing DNA vaccine immunogenicity and provided a basis for further investigation on their mutual effects on adjuvant activity and immune regulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Visual detection of glial cell line-derived neurotrophic factor based on a molecular translator and isothermal strand-displacement polymerization reaction.

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Zhang, Li-Yong; Xing, Tao; Du, Li-Xin; Li, Qing-Min; Liu, Wei-Dong; Wang, Ji-Yue; Cai, Jing

2015-01-01

Glial cell line-derived neurotrophic factor (GDNF) is a small protein that potently promotes the survival of many types of neurons. Detection of GDNF is vital to monitoring the survival of sympathetic and sensory neurons. However, the specific method for GDNF detection is also un-discovered. The purpose of this study is to explore the method for protein detection of GDNF. A novel visual detection method based on a molecular translator and isothermal strand-displacement polymerization reaction (ISDPR) has been proposed for the detection of GDNF. In this study, a molecular translator was employed to convert the input protein to output deoxyribonucleic acid signal, which was further amplified by ISDPR. The product of ISDPR was detected by a lateral flow biosensor within 30 minutes. This novel visual detection method based on a molecular translator and ISDPR has very high sensitivity and selectivity, with a dynamic response ranging from 1 pg/mL to 10 ng/mL, and the detection limit was 1 pg/mL of GDNF. This novel visual detection method exhibits high sensitivity and selectivity, which is very simple and universal for GDNF detection to help disease therapy in clinical practice.

Adult rat bone marrow stromal cells express genes associated with dopamine neurons

International Nuclear Information System (INIS)

Kramer, Brian C.; Woodbury, Dale; Black, Ira B.

2006-01-01

An intensive search is underway to identify candidates to replace the cells that degenerate in Parkinson's disease (PD). To date, no suitable substitute has been found. We have recently found that adult rat bone marrow stromal cells (MSCs) can be induced to assume a neuronal phenotype in vitro. These findings may have particular relevance to the treatment of PD. We now report that adult MSCs express multiple dopaminergic genes, suggesting that they are potential candidates for cell therapy. Using RT-PCR, we have examined families of genes that are associated with the development and/or survival of dopaminergic neurons. MSCs transcribe a variety of dopaminergic genes including patched and smoothened (components of the Shh receptor), Gli-1 (downstream mediator of Shh), and Otx-1, a gene associated with formation of the mesencephalon during development. Furthermore, Shh treatment elicits a 1.5-fold increase in DNA synthesis in cultured MSCs, suggesting the presence of a functional Shh receptor complex. We have also found that MSCs transcribe and translate Nurr-1, a nuclear receptor essential for the development of dopamine neurons. In addition, MSCs express a variety of growth factor receptors including the glycosyl-phosphatidylinositol-anchored ligand-binding subunit of the GDNF receptor, GFRα1, as well as fibroblast growth factor receptors one and four. The expression of genes that are associated with the development and survival of dopamine neurons suggests a potential role for these cells in the treatment of Parkinson's disease

Urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression

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Grether-Beck, Susanne; Felsner, Ingo; Brenden, Heidi; Kohne, Zippora; Majora, Marc; Marini, Alessandra; Jaenicke, Thomas; Rodriguez-Martin, Marina; Trullas, Carles; Hupe, Melanie; Elias, Peter M.; Krutmann, Jean

2012-01-01

Urea is an endogenous metabolite, known to enhance stratum corneum hydration. Yet, topical urea anecdotally also improves permeability barrier function, and it appears to exhibit antimicrobial activity. Hence, we hypothesized that urea is not merely a passive metabolite, but a small-molecule regulator of epidermal structure and function. In 21 human volunteers, topical urea improved barrier function in parallel with enhanced antimicrobial peptide (LL-37 and β-defensin-2) expression. Urea both stimulates expression of, and is transported into keratinocytes by two urea transporters, UT-A1 and UT-A2, and by aquaporin 3, 7 and 9. Inhibitors of these urea transporters block the downstream biological effects of urea, which include increased mRNA and protein levels for: (i) transglutaminase-1, involucrin, loricrin and filaggrin; (ii) epidermal lipid synthetic enzymes, and (iii) cathelicidin/LL-37 and β-defensin-2. Finally, we explored the potential clinical utility of urea, showing that topical urea applications normalized both barrier function and antimicrobial peptide expression in a murine model of atopic dermatitis (AD). Together, these results show that urea is a small-molecule regulator of epidermal permeability barrier function and antimicrobial peptide expression after transporter uptake, followed by gene regulatory activity in normal epidermis, with potential therapeutic applications in diseased skin. PMID:22418868

BAG3 increases the invasiveness of uterine corpus carcinoma cells by suppressing miR-29b and enhancing MMP2 expression.

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Habata, Shutaro; Iwasaki, Masahiro; Sugio, Asuka; Suzuki, Miwa; Tamate, Masato; Satohisa, Seiro; Tanaka, Ryoichi; Saito, Tsuyoshi

2015-05-01

Approximately 30% of uterine corpus carcinomas are diagnosed at an advanced stage and have a poor prognosis. Our previous study indicated that BCL2-associated athanogene 3 (BAG3) enhances matrix metalloproteinase-2 (MMP2) expression and binds to MMP2 to positively regulate the process of cell invasion in ovarian cancer cells. Recently, altered miRNA expression patterns were observed in several groups of patients with endometrial cancers. One of the altered miRNAs, miR-29b, reportedly reduces tumor invasiveness by suppressing MMP2 expression. Our aim in the present study was to examine the relationships among BAG3, miR-29b and MMP2 in endometrioid adenocarcinoma cells. We found that BAG3 suppresses miR-29b expression and enhances MMP2 expression, which in turn increases cell motility and invasiveness. Moreover, restoration of miR-29b through BAG3 knockdown reduced MMP2 expression, as well as cell motility and invasiveness. Collectively, our findings indicate that BAG3 enhances MMP2 expression by suppressing miR-29b, thereby increasing the metastatic potential of endometrioid adenocarcinomas.

Gamma knife irradiation of injured sciatic nerve induces histological and behavioral improvement in the rat neuropathic pain model.

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Yuki Yagasaki

Full Text Available We examined the effects of gamma knife (GK irradiation on injured nerves using a rat partial sciatic nerve ligation (PSL model. GK irradiation was performed at one week after ligation and nerve preparations were made three weeks after ligation. GK irradiation is known to induce immune responses such as glial cell activation in the central nervous system. Thus, we determined the effects of GK irradiation on macrophages using immunoblot and histochemical analyses. Expression of Iba-1 protein, a macrophage marker, was further increased in GK-treated injured nerves as compared with non-irradiated injured nerves. Immunohistochemical study of Iba-1 in GK-irradiated injured sciatic nerves demonstrated Iba-1 positive macrophage accumulation to be enhanced in areas distal to the ligation point. In the same area, myelin debris was also more efficiently removed by GK-irradiation. Myelin debris clearance by macrophages is thought to contribute to a permissive environment for axon growth. In the immunoblot study, GK irradiation significantly increased expressions of βIII-tubulin protein and myelin protein zero, which are markers of axon regeneration and re-myelination, respectively. Toluidine blue staining revealed the re-myelinated fiber diameter to be larger at proximal sites and that the re-myelinated fiber number was increased at distal sites in GK-irradiated injured nerves as compared with non-irradiated injured nerves. These results suggest that GK irradiation of injured nerves facilitates regeneration and re-myelination. In a behavior study, early alleviation of allodynia was observed with GK irradiation in PSL rats. When GK-induced alleviation of allodynia was initially detected, the expression of glial cell line-derived neurotrophic factor (GDNF, a potent analgesic factor, was significantly increased by GK irradiation. These results suggested that GK irradiation alleviates allodynia via increased GDNF. This study provides novel evidence that GK

Over-expression of histone H3K4 demethylase gene JMJ15 enhances salt tolerance in Arabidopsis

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Yuan eShen

2014-06-01

Full Text Available Histone H3 lysine 4 trimethylation (H3K4me3 has been shown to be involved in stress-responsive gene expression and gene priming in plants. However, the role of H3K4me3 resetting in the processes is not clear. In this work we studied the expression and function of Arabidopsis H3K4 demethylase gene JMJ15. We show that the expression of JMJ15 was relatively low and was limited to a number of tissues during vegetative growth but was higher in young floral organs. Over-expression of the gene in gain-of-function mutants reduced the plant height with accumulation of lignin in stems, while the loss-of-function mutation did not produce any visible phenotype. The gain-of-function mutants showed enhanced salt tolerance, whereas the loss-of-function mutant was more sensitive to salt compared to the wild type. Transcriptomic analysis revealed that over-expression of JMJ15 down-regulated many genes which are preferentially marked by H3K4me3 and H3K4me2. Many of the down-regulated genes encode transcription regulators involved in stress responses. The data suggest that increased JMJ15 levels may regulate the gene expression program that enhances stress tolerance.

60Co γ-irradiation enhances expression of GAP-43 mRNA in rat brain

International Nuclear Information System (INIS)

Su Bingyin; Cai Wenqin; Zhang Chenggang

2001-01-01

Objective: To study the relationship between the expression of GAP-43 mRNA and nerve regeneration in rat brain after 60 Co γ-irradiation. Methods: Wistar rats were subjected to whole-body irradiation with 8 Gy 60 Co γ-rays. The expression of GAP-43 was detected by in situ hybridization histochemistry using Dig-cRNA probe. Results: It was found that the expression of GAP-43 mRNA increased in the cerebral cortex, caudate, putamen, globus pallidum, thalamus and hypothalamus one week after 8 Gy 60 Co γ-irradiation. The peak of GAP-43 mRNA expression was observed in the fourth week and then began to decrease but still remained at a higher than normal level. However, it decreased to a low level after 7 weeks. Conclusion: Enhanced expression of GAP-43 mRNA after 60 Co γ-irradiation in rat brain is associated with nerve regeneration and reconstruction of synapse

Ethnicity moderates the outcomes of self-enhancement and self-improvement themes in expressive writing.

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Tsai, William; Lau, Anna S; Niles, Andrea N; Coello, Jordan; Lieberman, Matthew D; Ko, Ahra C; Hur, Christopher; Stanton, Annette L

2015-10-01

The current study examined whether writing content related to self-enhancing (viz., downward social comparison and situational attributions) and self-improving (viz., upward social comparison and persistence) motivations were differentially related to expressive writing outcomes among 17 Asian American and 17 European American participants. Content analysis of the essays revealed no significant cultural group differences in the likelihood of engaging in self-enhancing versus self-improving reflections on negative personal experiences. However, cultural group differences were apparent in the relation between self-motivation processes and changes in anxiety and depressive symptoms at 3-month follow-up. Among European Americans, writing that reflected downward social comparison predicted positive outcomes, whereas persistence writing themes were related to poorer outcomes. For Asian Americans, writing about persistence was related to positive outcomes, whereas downward social comparison and situational attributions predicted poorer outcomes. Findings provide evidence suggesting culturally distinct mechanisms for the effects of expressive disclosure. (PsycINFO Database Record (c) 2015 APA, all rights reserved).

The Impacts of Swimming Exercise on Hippocampal Expression of Neurotrophic Factors in Rats Exposed to Chronic Unpredictable Mild Stress

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Pei Jiang

2014-01-01

Full Text Available Depression is associated with stress-induced neural atrophy in limbic brain regions, whereas exercise has antidepressant effects as well as increasing hippocampal synaptic plasticity by strengthening neurogenesis, metabolism, and vascular function. A key mechanism mediating these broad benefits of exercise on the brain is induction of neurotrophic factors, which instruct downstream structural and functional changes. To systematically evaluate the potential neurotrophic factors that were involved in the antidepressive effects of exercise, in this study, we assessed the effects of swimming exercise on hippocampal mRNA expression of several classes of the growth factors (BDNF, GDNF, NGF, NT-3, FGF2, VEGF, and IGF-1 and peptides (VGF and NPY in rats exposed to chronic unpredictable mild stress (CUMS. Our study demonstrated that the swimming training paradigm significantly induced the expression of BDNF and BDNF-regulated peptides (VGF and NPY and restored their stress-induced downregulation. Additionally, the exercise protocol also increased the antiapoptotic Bcl-xl expression and normalized the CUMS mediated induction of proapoptotic Bax mRNA level. Overall, our data suggest that swimming exercise has antidepressant effects, increasing the resistance to the neural damage caused by CUMS, and both BDNF and its downstream neurotrophic peptides may exert a major function in the exercise related adaptive processes to CUMS.

Genistein enhances the effect of trichostatin A on inhibition of A549 cell growth by increasing expression of TNF receptor-1

International Nuclear Information System (INIS)

Wu, Tzu-Chin; Yang, Ying-Chihi; Huang, Pei-Ru; Wen, Yu-Der; Yeh, Shu-Lan

2012-01-01

Our previous study has shown that genistein enhances apoptosis in A549 lung cancer cells induced by trichostatin A (TSA). The precise molecular mechanism underlying the effect of genistein, however, remains unclear. In the present study, we investigated whether genistein enhances the anti-cancer effect of TSA through up-regulation of TNF receptor-1 (TNFR-1) death receptor signaling. We incubated A549 cells with TSA (50 ng/mL) alone or in combination with genistein and then determined the mRNA and protein expression of TNFR-1 as well as the activation of downstream caspases. Genistein at 5 and 10 μM significantly enhanced the TSA-induced decrease in cell number and apoptosis in a dose-dependent manner. The combined treatment significantly increased mRNA and protein expression of TNFR-1 at 6 and 12 h, respectively, compared with that of the control group; while TSA alone had no effect. TSA in combination with 10 μM of genistein increased TNFR-1 mRNA and protein expression by about 70% and 40%, respectively. The underlying mechanism for this effect of genistein may be partly associated with the estrogen receptor pathway. The combined treatment also increased the activation of caspase-3 and ‐10 as well as p53 protein expression in A549 cells. The enhancing effects of genistein on the TSA-induced decrease in cell number and on the expression of caspase-3 in A549 cells were suppressed by silencing TNFR-1 expression. These data demonstrated that the upregulation of TNFR-1 death receptor signaling plays an important role, at least in part, in the enhancing effect of genistein on TSA-induced apoptosis in A549 cells. -- Highlights: ► TSA combined with genistein rather than TSA alone increases the expression of TNFR-1. ► Genistein may exert such an effect partly through estrogen receptor pathway. ► The combined treatment increases the activation of caspase-10 and caspase-3. ► The combined treatment also increases the expression of p53 protein. ► TNFR-1 si

The Absence of NOD1 Enhances Killing of Aspergillus fumigatus Through Modulation of Dectin-1 Expression

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Mark S. Gresnigt

2017-12-01

Full Text Available One of the major life-threatening infections for which severely immunocompromised patients are at risk is invasive aspergillosis (IA. Despite the current treatment options, the increasing antifungal resistance and poor outcome highlight the need for novel therapeutic strategies to improve outcome of patients with IA. In the current study, we investigated whether and how the intracellular pattern recognition receptor NOD1 is involved in host defense against Aspergillus fumigatus. When exploring the role of NOD1 in an experimental mouse model, we found that Nod1−/− mice were protected against IA and demonstrated reduced fungal outgrowth in the lungs. We found that macrophages derived from bone marrow of Nod1−/− mice were more efficiently inducing reactive oxygen species and cytokines in response to Aspergillus. Most strikingly, these cells were highly potent in killing A. fumigatus compared with wild-type cells. In line, human macrophages in which NOD1 was silenced demonstrated augmented Aspergillus killing and NOD1 stimulation decreased fungal killing. The differentially altered killing capacity of NOD1 silencing versus NOD1 activation was associated with alterations in dectin-1 expression, with activation of NOD1 reducing dectin-1 expression. Furthermore, we were able to demonstrate that Nod1−/− mice have elevated dectin-1 expression in the lung and bone marrow, and silencing of NOD1 gene expression in human macrophages increases dectin-1 expression. The enhanced dectin-1 expression may be the mechanism of enhanced fungal killing of Nod1−/− cells and human cells in which NOD1 was silenced, since blockade of dectin-1 reversed the augmented killing in these cells. Collectively, our data demonstrate that NOD1 receptor plays an inhibitory role in the host defense against Aspergillus. This provides a rationale to develop novel immunotherapeutic strategies for treatment of aspergillosis that target the NOD1 receptor, to enhance the

Electroactive biodegradable polyurethane significantly enhanced Schwann cells myelin gene expression and neurotrophin secretion for peripheral nerve tissue engineering.

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Wu, Yaobin; Wang, Ling; Guo, Baolin; Shao, Yongpin; Ma, Peter X

2016-05-01

Myelination of Schwann cells (SCs) is critical for the success of peripheral nerve regeneration, and biomaterials that can promote SCs' neurotrophin secretion as scaffolds are beneficial for nerve repair. Here we present a biomaterials-approach, specifically, a highly tunable conductive biodegradable flexible polyurethane by polycondensation of poly(glycerol sebacate) and aniline pentamer, to significantly enhance SCs' myelin gene expression and neurotrophin secretion for peripheral nerve tissue engineering. SCs are cultured on these conductive polymer films, and the biocompatibility of these films and their ability to enhance myelin gene expressions and sustained neurotrophin secretion are successfully demonstrated. The mechanism of SCs' neurotrophin secretion on conductive films is demonstrated by investigating the relationship between intracellular Ca(2+) level and SCs' myelination. Furthermore, the neurite growth and elongation of PC12 cells are induced by adding the neurotrophin medium suspension produced from SCs-laden conductive films. These data suggest that these conductive degradable polyurethanes that enhance SCs' myelin gene expressions and sustained neurotrophin secretion perform great potential for nerve regeneration applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pre- and post-exposure safety and efficacy of attenuated rabies virus vaccines are enhanced by their expression of IFNγ

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Barkhouse, Darryll A. [Department of Cancer Biology, 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States); Center for Neurovirology 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States); Faber, Milosz [Center for Neurovirology 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States); Department of Microbiology and Immunology 1020 Locust St., Jefferson Alumni Hall, Room 465, Philadelphia, PA 19107 (United States); Hooper, D. Craig, E-mail: douglas.hooper@jefferson.edu [Department of Cancer Biology, 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States); Department of Neurological Surgery, 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States); Center for Neurovirology 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States)

2015-01-01

Consistent with evidence of a strong correlation between interferon gamma (IFNγ) production and rabies virus (RABV) clearance from the CNS, we recently demonstrated that engineering a pathogenic RABV to express IFNγ highly attenuates the virus. Reasoning that IFNγ expression by RABV vaccines would enhance their safety and efficacy, we reverse-engineered two proven vaccine vectors, GAS and GASGAS, to express murine IFNγ. Mortality and morbidity were monitored during suckling mice infection, immunize/challenge experiments and mixed intracranial infections. We demonstrate that GASγ and GASγGAS are significantly attenuated in suckling mice compared to the GASGAS vaccine. GASγ better protects mice from lethal DRV4 RABV infection in both pre- and post-exposure experiments compared to GASGAS. Finally, GASγGAS reduces post-infection neurological sequelae, compared to control, during mixed intracranial infection with DRV4. These data show IFNγ expression by a vaccine vector can enhance its safety while increasing its efficacy as pre- and post-exposure treatment. - Highlights: • IFNγ expression improves attenuated rabies virus safety and immunogenicity. • IFNγ expression is safer and more immunogenic than doubling glycoprotein expression. • Co-infection with IFNγ-expressing RABV prevents wild-type rabies virus lethality. • Vaccine safety and efficacy is additive for IFNγ and double glycoprotein expression.

Pre- and post-exposure safety and efficacy of attenuated rabies virus vaccines are enhanced by their expression of IFNγ

International Nuclear Information System (INIS)

Barkhouse, Darryll A.; Faber, Milosz; Hooper, D. Craig

2015-01-01

Consistent with evidence of a strong correlation between interferon gamma (IFNγ) production and rabies virus (RABV) clearance from the CNS, we recently demonstrated that engineering a pathogenic RABV to express IFNγ highly attenuates the virus. Reasoning that IFNγ expression by RABV vaccines would enhance their safety and efficacy, we reverse-engineered two proven vaccine vectors, GAS and GASGAS, to express murine IFNγ. Mortality and morbidity were monitored during suckling mice infection, immunize/challenge experiments and mixed intracranial infections. We demonstrate that GASγ and GASγGAS are significantly attenuated in suckling mice compared to the GASGAS vaccine. GASγ better protects mice from lethal DRV4 RABV infection in both pre- and post-exposure experiments compared to GASGAS. Finally, GASγGAS reduces post-infection neurological sequelae, compared to control, during mixed intracranial infection with DRV4. These data show IFNγ expression by a vaccine vector can enhance its safety while increasing its efficacy as pre- and post-exposure treatment. - Highlights: • IFNγ expression improves attenuated rabies virus safety and immunogenicity. • IFNγ expression is safer and more immunogenic than doubling glycoprotein expression. • Co-infection with IFNγ-expressing RABV prevents wild-type rabies virus lethality. • Vaccine safety and efficacy is additive for IFNγ and double glycoprotein expression

Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract.

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Connell, I; Agace, W; Klemm, P; Schembri, M; Mărild, S; Svanborg, C

1996-01-01

Type 1 fimbriae are adhesion organelles expressed by many Gram-negative bacteria. They facilitate adherence to mucosal surfaces and inflammatory cells in vitro, but their contribution to virulence has not been defined. This study presents evidence that type 1 fimbriae increase the virulence of Escherichia coli for the urinary tract by promoting bacterial persistence and enhancing the inflammatory response to infection. In a clinical study, we observed that disease severity was greater in chil...

AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

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Tian, Ying; Wang, Genjie; Hu, Qingzhu; Xiao, Xichun; Chen, Shuxia

2018-04-01

The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation. © 2017 Wiley Periodicals, Inc.

Neurotrophic factors and receptors in the immature and adult spinal cord after mechanical injury or kainic acid.

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Widenfalk, J; Lundströmer, K; Jubran, M; Brene, S; Olson, L

2001-05-15

Delivery of neurotrophic factors to the injured spinal cord has been shown to stimulate neuronal survival and regeneration. This indicates that a lack of sufficient trophic support is one factor contributing to the absence of spontaneous regeneration in the mammalian spinal cord. Regulation of the expression of neurotrophic factors and receptors after spinal cord injury has not been studied in detail. We investigated levels of mRNA-encoding neurotrophins, glial cell line-derived neurotrophic factor (GDNF) family members and related receptors, ciliary neurotrophic factor (CNTF), and c-fos in normal and injured spinal cord. Injuries in adult rats included weight-drop, transection, and excitotoxic kainic acid delivery; in newborn rats, partial transection was performed. The regulation of expression patterns in the adult spinal cord was compared with that in the PNS and the neonate spinal cord. After mechanical injury of the adult rat spinal cord, upregulations of NGF and GDNF mRNA occurred in meningeal cells adjacent to the lesion. BDNF and p75 mRNA increased in neurons, GDNF mRNA increased in astrocytes close to the lesion, and GFRalpha-1 and truncated TrkB mRNA increased in astrocytes of degenerating white matter. The relatively limited upregulation of neurotrophic factors in the spinal cord contrasted with the response of affected nerve roots, in which marked increases of NGF and GDNF mRNA levels were observed in Schwann cells. The difference between the ability of the PNS and CNS to provide trophic support correlates with their different abilities to regenerate. Kainic acid delivery led to only weak upregulations of BDNF and CNTF mRNA. Compared with several brain regions, the overall response of the spinal cord tissue to kainic acid was weak. The relative sparseness of upregulations of endogenous neurotrophic factors after injury strengthens the hypothesis that lack of regeneration in the spinal cord is attributable at least partly to lack of trophic support.

Butyrate transcriptionally enhances peptide transporter PepT1 expression and activity.

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Guillaume Dalmasso

Full Text Available BACKGROUND: PepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte. RESULTS: We found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1 promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by approximately 2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles. CONCLUSIONS: Collectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation.

MiR-143 enhances the antitumor activity of shikonin by targeting BAG3 expression in human glioblastoma stem cells.

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Liu, Jing; Qu, Cheng-Bin; Xue, Yi-Xue; Li, Zhen; Wang, Ping; Liu, Yun-hui

Therapeutic applications of microRNAs (miRNAs) in chemotherapy were confirmed to be valuable, but there is rare to identify their specific roles and functions in shikonin treatment toward tumors. Here, for the first time, we reported that miR-143 played a critical role in the antitumor activity of shikonin in glioblastoma stem cells (GSCs). The results showed that the expression of miR-143 was downregulated in shikonin treated GSCs within 24 h. MiR-143 overexpression significantly enhanced the inhibitory effect of shikonin toward GSCs on cell viability. Besides, miR-143 overexpression caused a significant increase in the apoptotic fraction and made apoptosis occur earlier. Further investigation identified that BAG3, an apoptotic regulator, was a functional target of miR-143 in shikonin treated GSCs. The expression of BAG3 was upregulated in shikonin treated GSCs within 24 h. MiR-143 overexpression significantly reversed the high expression of BAG3 in shikonin treated GSCs. Moreover, it was confirmed that the enhanced cytotoxicity of shikonin by miR-143 overexpression was reversed by BAG3 overexpression both in vitro and in vivo, suggesting that the enhanced tumor suppressive effects by miR-143 overexpression was at least partly through the regulation of BAG3. Taken together, for the first time, our results demonstrate that miR-143 could enhance the antitumor activity of shikonin toward GSCs through reducing BAG3 expression, which may provide a novel therapeutic strategy for enhancing the treatment efficacy of shikonin toward GSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent protein is not.

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Tao, Wen; Evans, Barbara-Graham; Yao, Jing; Cooper, Scott; Cornetta, Kenneth; Ballas, Christopher B; Hangoc, Giao; Broxmeyer, Hal E

2007-03-01

Validated gene transfer and expression tracers are essential for elucidating functions of mammalian genes. Here, we have determined the suitability and unintended side effects of enhanced green fluorescent protein (EGFP) and DsRed-Express fluorescent protein as expression tracers in long-term hematopoietic stem cells (HSCs). Retrovirally transduced mouse bone marrow cells expressing either EGFP or DsRed-Express in single or mixed dual-color cell populations were clearly discerned by flow cytometry and fluorescence microscopy. The results from in vivo competitive repopulation assays demonstrated that EGFP-expressing HSCs were maintained nearly throughout the lifespan of the transplanted mice and retained long-term multilineage repopulating potential. All mice assessed at 15 months post-transplantation were EGFP positive, and, on average, 24% total peripheral white blood cells expressed EGFP. Most EGFP-expressing recipient mice lived at least 22 months. In contrast, Discosoma sp. red fluorescent protein (DsRed)-expressing donor cells dramatically declined in transplant-recipient mice over time, particularly in the competitive setting, in which mixed EGFP- and DsRed-expressing cells were cotransplanted. Moreover, under in vitro culture condition favoring preservation of HSCs, purified EGFP-expressing cells grew robustly, whereas DsRed-expressing cells did not. Therefore, EGFP has no detectable deteriorative effects on HSCs, and is nearly an ideal long-term expression tracer for hematopoietic cells; however, DsRed-Express fluorescent protein is not suitable for these cells.

Expression of rabbit IL-4 by recombinant myxoma viruses enhances virulence and overcomes genetic resistance to myxomatosis.

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Kerr, P J; Perkins, H D; Inglis, B; Stagg, R; McLaughlin, E; Collins, S V; Van Leeuwen, B H

2004-06-20

Rabbit IL-4 was expressed in the virulent standard laboratory strain (SLS) and the attenuated Uriarra (Ur) strain of myxoma virus with the aim of creating a Th2 cytokine environment and inhibiting the development of an antiviral cell-mediated response to myxomatosis in infected rabbits. This allowed testing of a model for genetic resistance to myxomatosis in wild rabbits that have undergone 50 years of natural selection for resistance to myxomatosis. Expression of IL-4 significantly enhanced virulence of both virulent and attenuated virus strains in susceptible (laboratory) and resistant (wild) rabbits. SLS-IL-4 completely overcame genetic resistance in wild rabbits. The pathogenesis of SLS-IL-4 was compared in susceptible and resistant rabbits. The results support a model for resistance to myxomatosis of an enhanced innate immune response controlling virus replication and allowing an effective antiviral cell-mediated immune response to develop in resistant rabbits. Expression of IL-4 did not overcome immunity to myxomatosis induced by immunization.

Expression of an Arabidopsis Ca2+/H+ antiporter CAX1 variant in petunia enhances cadmium tolerance and accumulation.

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Wu, Qingyu; Shigaki, Toshiro; Williams, Kimberly A; Han, Jeung-Sul; Kim, Chang Kil; Hirschi, Kendal D; Park, Sunghun

2011-01-15

Phytoremediation is a cost-effective and minimally invasive technology to cleanse soils contaminated with heavy metals. However, few plant species are suitable for phytoremediation of metals such as cadmium (Cd). Genetic engineering offers a powerful tool to generate plants that can hyperaccumulate Cd. An Arabidopsis CAX1 mutant (CAXcd), which confers enhanced Cd transport in yeast, was ectopically expressed in petunia to evaluate whether the CAXcd expression would enhance Cd tolerance and accumulation in planta. The CAXcd-expressing petunia plants showed significantly greater Cd tolerance and accumulation than the controls. After being treated with either 50 or 100μM CdCl(2) for 6 weeks, the CAXcd-expressing plants showed more vigorous growth compared with controls, and the transgenic plants accumulated significantly more Cd (up to 2.5-fold) than controls. Moreover, the accumulation of Cd did not affect the development and morphology of the CAXcd-expressing petunia plants until the flowering and ultimately the maturing of seeds. Therefore, petunia has the potential to serve as a model species for developing herbaceous, ornamental plants for phytoremediation. Copyright © 2010 Elsevier GmbH. All rights reserved.

Microarray Gene Expression Analysis of Murine Tumor Heterogeneity Defined by Dynamic Contrast-Enhanced MRI

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Nick G. Costouros

2002-07-01

Full Text Available Current methods of studying angiogenesis are limited in their ability to serially evaluate in vivo function throughout a target tissue. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI and pharmacokinetic modeling provide a useful method for evaluating tissue vasculature based on contrast accumulation and washout. While it is often assumed that areas of high contrast enhancement and washout comprise areas of increased angiogenesis and tumor activity, the actual molecular pathways that are active in such areas are poorly understood. Using DCE-MRI in a murine subcutaneous tumor model, we were able to perform pharmacokinetic functional analysis of a tumor, coregistration of MRI images with histological cross-sections, immunohistochemistry, laser capture microdissection, and genetic profiling of tumor heterogeneity based on pharmacokinetic parameters. Using imaging as a template for biologic investigation, we have not found evidence of increased expression of proangiogenic modulators at the transcriptional level in either distinct pharmacokinetic region. Furthermore, these regions show no difference on histology and CD31 immunohistochemistry. However, the expression of ribosomal proteins was greatly increased in high enhancement and washout regions, implying increased protein translation and consequent increased cellular activity. Together, these findings point to the potential importance of posttranscriptional regulation in angiogenesis and the need for the development of angiogenesis-specific contrast agents to evaluate in vivo angiogenesis at a molecular level.

Expressive Suppression and Enhancement During Music-Elicited Emotions in Younger and Older Adults

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Sandrine eVieillard

2015-02-01

Full Text Available When presented with emotional visual scenes, older adults have been found to be equally capable to regulate emotion expression as younger adults, corroborating the view that emotion regulation skills are maintained or even improved in later adulthood. However, the possibility that gaze direction might help achieve an emotion control goal has not been taken into account, raising the question whether the effortful processing of expressive regulation is really spared from the general age-related decline. Since it does not allow perceptual attention to be redirected away from the emotional source, music provides a useful way to address this question. In the present study, affective, behavioral and physiological consequences of free expression of emotion, expressive suppression and expressive enhancement were measured in 31 younger and 30 older adults while they listened to positive and negative musical excerpts. The main results indicated that compared to younger adults, older adults reported experiencing less emotional intensity in response to negative music during the free expression of emotion condition. No age difference was found in the ability to amplify or reduce emotional expressions. However, an age-related decline in the ability to reduce the intensity of emotional state and an age-related increase in physiological reactivity were found when participants were instructed to suppress negative expression. Taken together, the current data support previous findings suggesting an age-related change in response to music. They also corroborate the observation that older adults are as efficient as younger adults at controlling behavioral expression. But most importantly, they suggest that when faced with auditory sources of negative emotion, older age does not always confer a better ability to regulate emotions.

Activated microglia induce bone marrow mesenchymal stem cells to produce glial cell-derived neurotrophic factor and protect neurons against oxygen-glucose deprivation injury

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Bingke Lv

2016-12-01

Full Text Available In this study, we investigated interactions among microglia (MG, bone marrow mesenchymal stem cells (BMSCs and neurons in cerebral ischemia and the potential mechanisms using an in vitro oxygen-glucose deprivation (OGD model. Rat BMSCs were incubated with conditioned medium (CM from in vitro cultures of OGD-activated rat MG and murine BV2 MG cells. Effects of glial cell-derived neurotrophic factor (GDNF on rat neuron viability, apoptosis, lactate dehydrogenase (LDH leakage and mitochondrial membrane potential (MMP were analyzed in this model. OGD-activated MG promoted GDNF production by BMSCs (P < 0.01. TNFα, but not IL6 or IL1β, promoted GDNF production by BMSCs (P < 0.001. GDNF or CM pre-treated BMSCs elevated neuronal viability and suppressed apoptosis (P < 0.05 or P < 0.01; these effects were inhibited by the RET antibody. GDNF activated MEK/ERK and PI3K/AKT signaling but not JNK/c-JUN. Furthermore, GDNF upregulated B cell lymphoma 2 (BCL2 and heat shock 60 kDa protein 1 (HSP60 levels, suppressed LDH leakage, and promoted MMP. Thus, activated MG produce TNFα to stimulate GDNF production by BMSCs, which prevents and repairs OGD-induced neuronal injury, possibly via regulating MEK/ERK and PI3K/AKT signaling. These findings will facilitate the prevention and treatment of neuronal injury by cerebral ischemia.

Suppression of LFA-1 expression by spermine is associated with enhanced methylation of ITGAL, the LFA-1 promoter area.

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Yoshihiko Kano

Full Text Available Spermine and spermidine, natural polyamines, suppress lymphocyte function-associated antigen 1 (LFA-1 expression and its associated cellular functions through mechanisms that remain unknown. Inhibition of ornithine decarboxylase, which is required for polyamine synthesis, in Jurkat cells by 3 mM D,L-alpha-difluoromethylornithine hydrochloride (DFMO significantly decreased spermine and spermidine concentrations and was associated with decreased DNA methyltransferase (Dnmt activity, enhanced demethylation of the LFA-1 gene (ITGAL promoter area, and increased CD11a expression. Supplementation with extracellular spermine (500 µM of cells pretreated with DFMO significantly increased polyamine concentrations, increased Dnmt activity, enhanced methylation of the ITGAL promoter, and decreased CD11a expression. It has been shown that changes in intracellular polyamine concentrations affect activities of -adenosyl-L-methionine-decaroboxylase, and, as a result, affect concentrations of the methyl group donor, S-adenosylmethionine (SAM, and of the competitive Dnmt inhibitor, decarboxylated SAM. Additional treatments designed to increase the amount of SAM and decrease the amount of decarboxylated SAM-such as treatment with methylglyoxal bis-guanylhydrazone (an inhibitor of S-adenosyl-L-methionine-decaroboxylase and SAM supplementation-successfully decreased CD11a expression. Western blot analyses revealed that neither DFMO nor spermine supplementation affected the amount of active Ras-proximate-1, a member of the Ras superfamily of small GTPases and a key protein for regulation of CD11a expression. The results of this study suggest that polyamine-induced suppression of LFA-1 expression occurs via enhanced methylation of ITGAL.

Carbon monoxide may enhance bile secretion by increasing glutathione excretion and Mrp2 expression in rats

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Chiung-Yu Chen

2013-05-01

Conclusion: The present study demonstrated that CO enhanced biliary output in conjunction with NO by increasing the biliary excretion of glutathione. The increment in biliary glutathione was associated with an increased expression of hepatic Mrp2.

Ectopic Expression of JcWRKY Confers Enhanced Resistance in Transgenic Tobacco Against Macrophomina phaseolina.

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Agarwal, Parinita; Patel, Khantika; Agarwal, Pradeep K

2018-04-01

Plants possess an innate immune system comprising of a complex network of closely regulated defense responses involving differential gene expression mediated by transcription factors (TFs). The WRKYs comprise of an important plant-specific TF family, which is involved in regulation of biotic and abiotic defenses. The overexpression of JcWRKY resulted in improved resistance in transgenic tobacco against Macrophomina phaseolina. The production of reactive oxygen species (ROS) and its detoxification through antioxidative system in the transgenics facilitates defense against Macrophomina. The enhanced catalase activity on Macrophomina infection limits the spread of infection. The transcript expression of antioxidative enzymes gene (CAT and SOD) and salicylic acid (SA) biosynthetic gene ICS1 showed upregulation during Macrophomina infection and combinatorial stress. The enhanced transcript of pathogenesis-related genes PR-1 indicates the accumulation of SA during different stresses. The PR-2 and PR-5 highlight the activation of defense responses comprising of activation of hydrolytic cleavage of glucanases and thaumatin-like proteins causing disruption of fungal cells. The ROS homeostasis in coordination with signaling molecules regulate the defense responses and inhibit fungal growth.

Cytomegalovirus vector expressing RAE-1γ induces enhanced anti-tumor capacity of murine CD8+ T cells.

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Tršan, Tihana; Vuković, Kristina; Filipović, Petra; Brizić, Ana Lesac; Lemmermann, Niels A W; Schober, Kilian; Busch, Dirk H; Britt, William J; Messerle, Martin; Krmpotić, Astrid; Jonjić, Stipan

2017-08-01

Designing CD8 + T-cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE-1γ as CD8 + T cell-based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE-1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD-1. CMV vector expressing RAE-1γ potentiated expansion of KLRG1 + CD8 + T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long-term maintenance of epitope-specific CD8 + T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8 + T-cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8 + T-cell sensitive tumors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expanding the Repertoire of Optogenetically Targeted Cells with an Enhanced Gene Expression System

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Kenji F. Tanaka

2012-08-01

Full Text Available Optogenetics has been enthusiastically pursued in recent neuroscience research, and the causal relationship between neural activity and behavior is becoming ever more accessible. Here, we established knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet and succeeded in generating transgenic mice expressing a highly light-sensitive channelrhodopsin-2 mutant at levels sufficient to drive the activities of multiple cell types. This method requires two lines of mice: one that controls the pattern of expression and another that determines the protein to be produced. The generation of new lines of either type readily expands the repertoire to choose from. In addition to neurons, we were able to manipulate the activity of nonexcitable glial cells in vivo. This shows that our system is applicable not only to neuroscience but also to any biomedical study that requires understanding of how the activity of a selected population of cells propagates through the intricate organic systems.

Identification of an enhancer that increases miR-200b~200a~429 gene expression in breast cancer cells.

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Joanne L Attema

Full Text Available The miR-200b~200a~429 gene cluster is a key regulator of EMT and cancer metastasis, however the transcription-based mechanisms controlling its expression during this process are not well understood. We have analyzed the miR-200b~200a~429 locus for epigenetic modifications in breast epithelial and mesenchymal cell lines using chromatin immunoprecipitation assays and DNA methylation analysis. We discovered a novel enhancer located approximately 5.1kb upstream of the miR-200b~200a~429 transcriptional start site. This region was associated with the active enhancer chromatin signature comprising H3K4me1, H3K27ac, RNA polymerase II and CpG dinucleotide hypomethylation. Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated miR-200b eRNA. Over-expression of miR-200b eRNA had little effect on miR-200b~200a~429 promoter activity and its production did not correlate with miR-200b~200a~429 gene expression. While additional investigations of miR-200b eRNA function will be necessary, it is possible that miR-200b eRNA may be involved in the regulation of miR-200b~200a~429 gene expression and silencing. Taken together, these findings reveal the presence of a novel enhancer, which contributes to miR-200b~200a~429 transcriptional regulation in epithelial cells.

Salinomycin enhances cisplatin-induced cytotoxicity in human lung cancer cells via down-regulation of AKT-dependent thymidylate synthase expression.

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Ko, Jen-Chung; Zheng, Hao-Yu; Chen, Wen-Ching; Peng, Yi-Shuan; Wu, Chia-Hung; Wei, Chia-Li; Chen, Jyh-Cheng; Lin, Yun-Wei

2016-12-15

Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2μg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

The dependence receptor Ret induces apoptosis in somatotrophs through a Pit-1/p53 pathway, preventing tumor growth.

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Cañibano, Carmen; Rodriguez, Noela L; Saez, Carmen; Tovar, Sulay; Garcia-Lavandeira, Montse; Borrello, Maria Grazia; Vidal, Anxo; Costantini, Frank; Japon, Miguel; Dieguez, Carlos; Alvarez, Clara V

2007-04-18

Somatotrophs are the only pituitary cells that express Ret, GFRalpha1 and GDNF. This study investigated the effects of Ret in a somatotroph cell line, in primary pituitary cultures and in Ret KO mice. Ret regulates somatotroph numbers by inducing Pit-1 overexpression, leading to increased p53 expression and apoptosis, both of which can be prevented with Ret or Pit-1 siRNA. The Pit-1 overexpression is mediated by sustained activation of PKCdelta, JNK, c/EBPalpha and CREB induced by a complex of Ret, caspase 3 and PKCdelta. In the presence of GDNF, Akt is activated, and the Pit-1 overexpression and resulting apoptosis are blocked. The adenopituitary of Ret KO mice is larger than normal, showing Pit-1 and somatotroph hyperplasia. In normal animals, activation of the Ret/Pit-1/p53 pathway by retroviral introduction of Ret blocked tumor growth in vivo. Thus, somatotrophs have an intrinsic mechanism for controlling Pit-1/GH production through an apoptotic/survival pathway. Ret might be of value for treatment of pituitary adenomas.

Expression of ethylene biosynthetic and receptor genes in rose floral tissues during ethylene-enhanced flower opening

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Xue, Jingqi; Li, Yunhui; Tan, Hui; Yang, Feng; Ma, Nan; Gao, Junping

2008-01-01

Ethylene production, as well as the expression of ethylene biosynthetic (Rh-ACS1?4 and Rh-ACO1) and receptor (Rh-ETR1?5) genes, was determined in five different floral tissues (sepals, petals, stamens, gynoecia, and receptacles) of cut rose (Rosa hybrida cv. Samantha upon treatment with ethylene or the ethylene inhibitor 1-methylcyclopropene (1-MCP). Ethylene-enhanced ethylene production occurred only in gynoecia, petals, and receptacles, with gynoecia showing the greatest enhancement in the ...

Protective effects of two constituents of Chinese herbs on spinal motor neurons from embryonic rats with hypoxia injury.

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Chen, Jian-Feng; Fan, Jian; Tian, Xiao-Wu; Tang, Tian-Si

2012-01-01

Neuroprotective agents are becoming significant tools in the repair of central nervous system injuries. In this study, we determined whether ginkgolides (Gin, extract of GinkgoBiloba) and Acanthopanax senticosus saponins (ASS, flavonoids extracted from Acanthopanax herbal preparations) have protective effects on rat spinal cords exposed to anoxia and we explored the mechanisms that underlie the protective effects. Spinal motor neurons (SMNs) from rat spinal cords were obtained and divided into five groups with 10 wells in each group. In control group, SMNs suffered no injury under normal oxygen; in hypoxia- inducible (HI) group, SMNs suffered injury from hypoxia; in Gin group, 37.5µg/ml Gin were used before 24 hrs of hypoxia; in ASS group, 50µg/ml ASS were used before 24 hrs of hypoxia;in glial cell-lined derived neurotrophic factor (GDNF) group, 0.1µg/ml GDNF were used before 24 hrs of hypoxia. Changes in morphology, neuron viability, and lactate dehydrogenase (LDH) release were observed. In addition, the expression of HIF-1α induced by hypoxia was measured. The neuronal viability in the Gin, ASS, and GDNF pretreated groups was higher than that in the HI group (P0.05). The quantity of LDH released in the three pretreated groups was lower than that in the HI group (Phypoxic neurons.

Effect of enhanced expression of connexin 43 on sunitinib-induced cytotoxicity in mesothelioma cells

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Miaki Uzu

2015-05-01

Full Text Available Connexin (Cx makes up a type of intercellular channel called gap junction (GJ. GJ plays a regulatory role in cellular physiology. The Cx expression level is often decreased in cancer cells compared to that in healthy ones, and the restoration of its expression has been shown to exert antiproliferative effects. This work aims to evaluate the effect of the restoration of connexin 43 (Cx43 (the most ubiquitous Cx subtype expression on sunitinib (SU-induced cytotoxicity in malignant mesothelioma (MM cells. Increased Cx43 expression in an MM cell line (H28 improved the ability of SU to inhibit receptor tyrosine kinase (RTK signaling. Moreover, higher Cx43 expression promoted SU-induced apoptosis. The cell viability test revealed that Cx43 enhanced the cytotoxic effect of SU in a GJ-independent manner. The effect of Cx43 on a proapoptotic factor, Bax, was then investigated. The interaction between Cx43 and Bax was confirmed by immunoprecipitation. Furthermore, higher Cx43 expression increased the production of a cleaved (active form of Bax during SU-induced apoptosis with no alteration in total Bax expression. These findings indicate that Cx43 most likely increases sensitivity to SU in H28 through direct interaction with Bax. In conclusion, we found that Cx43 overcame the chemoresistance of MM cells.

Encapsulated cell device approach for combined electrical stimulation and neurotrophic treatment of the deaf cochlea.

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Konerding, W S; Janssen, H; Hubka, P; Tornøe, J; Mistrik, P; Wahlberg, L; Lenarz, T; Kral, A; Scheper, V

2017-07-01

Profound hearing impairment can be overcome by electrical stimulation (ES) of spiral ganglion neurons (SGNs) via a cochlear implant (CI). Thus, SGN survival is critical for CI efficacy. Application of glial cell line-derived neurotrophic factor (GDNF) has been shown to reduce SGN degeneration following deafness. We tested a novel method for local, continuous GDNF-delivery in combination with ES via a CI. The encapsulated cell (EC) device contained a human ARPE-19 cell-line, genetically engineered for secretion of GDNF. In vitro, GDNF delivery was stable during ES delivered via a CI. In the chronic in vivo part, cats were systemically deafened and unilaterally implanted into the scala tympani with a CI and an EC device, which they wore for six months. The implantation of control devices (same cell-line not producing GDNF) had no negative effect on SGN survival. GDNF application without ES led to an unexpected reduction in SGN survival, however, the combination of GDNF with initial, short-term ES resulted in a significant protection of SGNs. A tight fibrous tissue formation in the scala tympani of the GDNF-only group is thought to be responsible for the increased SGN degeneration, due to mechanisms related to an aggravated foreign body response. Furthermore, the fibrotic encapsulation of the EC device led to cell death or cessation of GDNF release within the EC device during the six months in vivo. In both in vitro and in vivo, fibrosis was reduced by CI stimulation, enabling the neuroprotective effect of the combined treatment. Thus, fibrous tissue growth limits treatment possibilities with an EC device. For a stable and successful long-term neurotrophic treatment of the SGN via EC devices in human CI users, it would be necessary to make changes in the treatment approach (provision of anti-inflammatories), the EC device surface (reduced cell adhesion) and the ES (initiation prior to fibrosis formation). Copyright © 2017 Elsevier B.V. All rights reserved.

Enhanced expression of two discrete isoforms of matrix metalloproteinase-2 in experimental and human diabetic nephropathy.

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Sang Soo Kim

Full Text Available We recently reported on the enhanced expression of two isoforms of matrix metalloproteinase-2 (MMP-2 in human renal transplantation delayed graft function. These consist of the conventional secreted, full length MMP-2 isoform (FL-MMP-2 and a novel intracellular N-Terminal Truncated isoform (NTT-MMP-2 generated by oxidative stress-mediated activation of an alternate promoter in the MMP-2 first intron. Here we evaluated the effect of hyperglycemia and diabetes mellitus on the in vitro and in vivo expression of the two MMP-2 isoforms.We quantified the abundance of the FL-MMP-2 and NTT-MMP-2 transcripts by qPCR in HK2 cells cultured in high glucose or 4-hydroxy-2-hexenal (HHE and tested the effects of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC. The streptozotocin (STZ murine model of Type I diabetes mellitus and renal biopsies of human diabetic nephropathy were used in this study.Both isoforms of MMP-2 in HK2 cells were upregulated by culture in high glucose or with HHE. PDTC treatment did not suppress high glucose-mediated FL-MMP-2 expression but potently inhibited NTT-MMP-2 expression. With STZ-treated mice, renal cortical expression of both isoforms was increased (FL-MMP-2, 1.8-fold; NTT-MMP-2, greater than 7-fold. Isoform-specific immunohistochemical staining revealed low, but detectable levels of the FL-MMP-2 isoform in controls, while NTT-MMP-2 was not detected. While there was a modest increase in tubular epithelial cell staining for FL-MMP-2 in STZ-treated mice, NTT-MMP-2 was intensely expressed in a basolateral pattern. FL-MMP-2 and NTT-MMP-2 isoform expression as quantified by qPCR were both significantly elevated in renal biopsies of human diabetic nephropathy (12-fold and 3-fold, respectively.The expression of both isoforms of MMP-2 was enhanced in an experimental model of diabetic nephropathy and in human diabetic nephropathy. Selective MMP-2 isoform inhibition could offer a novel approach for the treatment of diabetic renal

Selectively enhanced expression of prophenoloxidase activating enzyme 1 (PPAE1 at a bacteria clearance site in the white shrimp, Litopenaeus vannamei

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Jang In-Kwon

2011-12-01

Full Text Available Abstract Background The prophenoloxidase-activating (PO activating system plays an important role in the crustacean innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating enzyme (PPAE, which is the direct activator of prophenoloxidase (proPO. Despite their importance in crustacean PO activating system, the studies on them remain limited. Results Here we report on a PPAE of white shrimp, Litopenaeus vannamei (lvPPAE1, which showed 94% similarity to PPAE1 of Penaeus monodon. We found that lvPPAE1 in fluid hemocytes was down regulated after challenge by Vibrio harveyi but was enhanced when shrimps were exposed to a bacteria-rich environment for long-term. In vivo gene silence of lvPPAE1 by RNAi can significantly reduce the phenoloxidase activity (PO and increase the susceptibility of shrimps to V. harveyi. Although lvPPAE1 was down-regulated in fluid hemocytes by Vibrio challenge, its expression increased significantly in gill after bacteria injection, which is the primary bacteria-clearance tissue. Conclusion Suppressed expression in fluid hemocytes and enhanced expression in gill indicates selectively enhanced expression at the bacterial clearance site. This is a novel feature for PPAE expression. The results will contribute to our understanding of the PO activating system in crustaceans.

Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

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Maggi, Maristella; Scotti, Claudia

2017-08-01

Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

Zcchc11 Uridylates Mature miRNAs to Enhance Neonatal IGF-1 Expression, Growth, and Survival

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Kozlowski, Elyse; Matsuura, Kori Y.; Ferrari, Joseph D.; Morris, Samantha A.; Powers, John T.; Daley, George Q.; Quinton, Lee J.; Mizgerd, Joseph P.

2012-01-01

The Zcchc11 enzyme is implicated in microRNA (miRNA) regulation. It can uridylate let-7 precursors to decrease quantities of the mature miRNA in embryonic stem cell lines, suggested to mediate stem cell maintenance. It can uridylate mature miR-26 to relieve silencing activity without impacting miRNA content in cancer cell lines, suggested to mediate cytokine and growth factor expression. Broader roles of Zcchc11 in shaping or remodeling the miRNome or in directing biological or physiological processes remain entirely speculative. We generated Zcchc11-deficient mice to address these knowledge gaps. Zcchc11 deficiency had no impact on embryogenesis or fetal development, but it significantly decreased survival and growth immediately following birth, indicating a role for this enzyme in early postnatal fitness. Deep sequencing of small RNAs from neonatal livers revealed roles of this enzyme in miRNA sequence diversity. Zcchc11 deficiency diminished the lengths and terminal uridine frequencies for diverse mature miRNAs, but it had no influence on the quantities of any miRNAs. The expression of IGF-1, a liver-derived protein essential to early growth and survival, was enhanced by Zcchc11 expression in vitro, and miRNA silencing of IGF-1 was alleviated by uridylation events observed to be Zcchc11-dependent in the neonatal liver. In neonatal mice, Zcchc11 deficiency significantly decreased IGF-1 mRNA in the liver and IGF-1 protein in the blood. We conclude that the Zcchc11-mediated terminal uridylation of mature miRNAs is pervasive and physiologically significant, especially important in the neonatal period for fostering IGF-1 expression and enhancing postnatal growth and survival. We propose that the miRNA 3′ terminus is a regulatory node upon which multiple enzymes converge to direct silencing activity and tune gene expression. PMID:23209448

Zcchc11 uridylates mature miRNAs to enhance neonatal IGF-1 expression, growth, and survival.

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Matthew R Jones

Full Text Available The Zcchc11 enzyme is implicated in microRNA (miRNA regulation. It can uridylate let-7 precursors to decrease quantities of the mature miRNA in embryonic stem cell lines, suggested to mediate stem cell maintenance. It can uridylate mature miR-26 to relieve silencing activity without impacting miRNA content in cancer cell lines, suggested to mediate cytokine and growth factor expression. Broader roles of Zcchc11 in shaping or remodeling the miRNome or in directing biological or physiological processes remain entirely speculative. We generated Zcchc11-deficient mice to address these knowledge gaps. Zcchc11 deficiency had no impact on embryogenesis or fetal development, but it significantly decreased survival and growth immediately following birth, indicating a role for this enzyme in early postnatal fitness. Deep sequencing of small RNAs from neonatal livers revealed roles of this enzyme in miRNA sequence diversity. Zcchc11 deficiency diminished the lengths and terminal uridine frequencies for diverse mature miRNAs, but it had no influence on the quantities of any miRNAs. The expression of IGF-1, a liver-derived protein essential to early growth and survival, was enhanced by Zcchc11 expression in vitro, and miRNA silencing of IGF-1 was alleviated by uridylation events observed to be Zcchc11-dependent in the neonatal liver. In neonatal mice, Zcchc11 deficiency significantly decreased IGF-1 mRNA in the liver and IGF-1 protein in the blood. We conclude that the Zcchc11-mediated terminal uridylation of mature miRNAs is pervasive and physiologically significant, especially important in the neonatal period for fostering IGF-1 expression and enhancing postnatal growth and survival. We propose that the miRNA 3' terminus is a regulatory node upon which multiple enzymes converge to direct silencing activity and tune gene expression.

Infection-Induced Retrotransposon-Derived Noncoding RNAs Enhance Herpesviral Gene Expression via the NF-κB Pathway.

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John Karijolich

Full Text Available Short interspersed nuclear elements (SINEs are highly abundant, RNA polymerase III-transcribed noncoding retrotransposons that are silenced in somatic cells but activated during certain stresses including viral infection. How these induced SINE RNAs impact the host-pathogen interaction is unknown. Here we reveal that during murine gammaherpesvirus 68 (MHV68 infection, rapidly induced SINE RNAs activate the antiviral NF-κB signaling pathway through both mitochondrial antiviral-signaling protein (MAVS-dependent and independent mechanisms. However, SINE RNA-based signaling is hijacked by the virus to enhance viral gene expression and replication. B2 RNA expression stimulates IKKβ-dependent phosphorylation of the major viral lytic cycle transactivator protein RTA, thereby enhancing its activity and increasing progeny virion production. Collectively, these findings suggest that SINE RNAs participate in the innate pathogen response mechanism, but that herpesviruses have evolved to co-opt retrotransposon activation for viral benefit.

Enhanced salt stress tolerance in transgenic potato plants expressing IbMYB1, a sweet potato transcription factor.

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Cheng, Yu-Jie; Kim, Myoung-Duck; Deng, Xi-Ping; Kwak, Sang-Soo; Chen, Wei

2013-12-01

IbMYB1, a transcription factor (TF) for R2R3-type MYB TFs, is a key regulator of anthocyanin biosynthesis during storage of sweet potatoes. Anthocyanins provide important antioxidants of nutritional value to humans, and also protect plants from oxidative stress. This study aimed to increase transgenic potatoes' (Solanum tuberosum cv. LongShu No.3) tolerance to environmental stress and enhance their nutritional value. Transgenic potato plants expressing IbMYB1 genes under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SM plants) were successfully generated through Agrobacterium-mediated transformation. Two representative transgenic SM5 and SM12 lines were evaluated for enhanced tolerance to salinity, UV-B rays, and drought conditions. Following treatment of 100 mM NaCl, seedlings of SM5 and SM12 lines showed less root damage and more shoot growth than control lines expressing only an empty vector. Transgenic potato plants in pots treated with 400 mM NaCl showed high amounts of secondary metabolites, including phenols, anthocyanins, and flavonoids, compared with control plants. After treatment of 400 mM NaCl, transgenic potato plants also showed high DDPH radical scavenging activity and high PS II photochemical efficiency compared with the control line. Furthermore, following treatment of NaCl, UV-B, and drought stress, the expression levels of IbMYB1 and several structural genes in the flavonoid biosynthesis such as CHS, DFR, and ANS in transgenic plants were found to be correlated with plant phenotype. The results suggest that enhanced IbMYB1 expression affects secondary metabolism, which leads to improved tolerance ability in transgenic potatoes.

CCAAT/enhancer binding protein a gene expression in Egyptian patients with acute myeloid leukemia

International Nuclear Information System (INIS)

Kassem, N.; Fahmy, A.; Desoky, M.; Zawam, H.M.; Medhat, N.; Medhat, N.

2013-01-01

Background: Transcription factors play a crucial role in myeloid differentiation and lineage determination. Tumor suppressor protein C/EBPa is a key regulator of granulocytic differentiation whose functional inactivation has become a pathophysiological signature of myeloid leukemia. Given the role that CCAAT/enhancer binding protein α (C/EBP α) plays in myelopoiesis, we anticipated that their expression might be disrupted in myeloid neoplasms. Purpose: To estimate the expression of C/EBP α mRNA in patients with acute myeloid leukemia and correlate its expression with the pathogenesis of the disease. Patients and methods: Forty AML patients and 20 age and sex matched healthy controls were included in the study. Blood samples of patients and controls were analyzed for CEBP α mRNA expression by quantitative RT-real time PCR using TaqMan technology and δδct method for calculation of gene expression. Results: Twenty-nine (72.5%) patients out of the 40 showed low expression levels of CEBP α mRNA below the cutoff value with median of 0.19 (range:0-0.87). While eleven (27.5%) patients out of the 40 showed higher expression levels of CEBP α above the cutoff value with median of 1.52 (range: 1.07-2). Seven patients out of the 11 showed higher expression levels of CEBP α mRNA belong to the M3 subtype of AML harboring the t(15;17) PML-RARa translocation. Conclusion: We conclude that the majority of the AML patients analyzed, express low levels of C/EBPa mRN. However, a subset of patients represented by the M3 subtype, express higher levels of C/EBPa

Reconstruction of mouse testicular cellular microenvironments in long-term seminiferous tubule culture.

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Juho-Antti Mäkelä

Full Text Available Research on spermatogonia is hampered by complex architecture of the seminiferous tubule, poor viability of testicular tissue ex vivo and lack of physiologically relevant long-term culture systems. Therefore there is a need for an in vitro model that would enable long term survival and propagation of spermatogonia. We aimed at the most simplified approach to enable all different cell types within the seminiferous tubules to contribute to the creation of a niche for spermatogonia. In the present study we describe the establishment of a co-culture of mouse testicular cells that is based on proliferative and migratory activity of seminiferous tubule cells and does not involve separation, purification or differential plating of individual cell populations. The co-culture is composed of the constituents of testicular stem cell niche: Sertoli cells [identified by expression of Wilm's tumour antigen 1 (WT1 and secretion of glial cell line-derived neurotrophic factor, GDNF], peritubular myoid cells (expressing alpha smooth muscle actin, αSMA and spermatogonia [expressing MAGE-B4, PLZF (promyelocytic leukaemia zinc finger, LIN28, Gpr125 (G protein-coupled receptor 125, CD9, c-Kit and Nanog], and can be maintained for at least five weeks. GDNF was found in the medium at a sufficient concentration to support proliferating spermatogonial stem cells (SSCs that were able to start spermatogenic differentiation after transplantation to an experimentally sterile recipient testis. Gdnf mRNA levels were elevated by follicle-stimulating hormone (FSH which shows that the Sertoli cells in the co-culture respond to physiological stimuli. After approximately 2-4 weeks of culture a spontaneous formation of cord-like structures was monitored. These structures can be more than 10 mm in length and branch. They are formed by peritubular myoid cells, Sertoli cells, fibroblasts and spermatogonia as assessed by gene expression profiling. In conclusion, we have managed to

Duplicated Enhancer Region Increases Expression of CTSB and Segregates with Keratolytic Winter Erythema in South African and Norwegian Families.

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Ngcungcu, Thandiswa; Oti, Martin; Sitek, Jan C; Haukanes, Bjørn I; Linghu, Bolan; Bruccoleri, Robert; Stokowy, Tomasz; Oakeley, Edward J; Yang, Fan; Zhu, Jiang; Sultan, Marc; Schalkwijk, Joost; van Vlijmen-Willems, Ivonne M J J; von der Lippe, Charlotte; Brunner, Han G; Ersland, Kari M; Grayson, Wayne; Buechmann-Moller, Stine; Sundnes, Olav; Nirmala, Nanguneri; Morgan, Thomas M; van Bokhoven, Hans; Steen, Vidar M; Hull, Peter R; Szustakowski, Joseph; Staedtler, Frank; Zhou, Huiqing; Fiskerstrand, Torunn; Ramsay, Michele

2017-05-04

Keratolytic winter erythema (KWE) is a rare autosomal-dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling. KWE was previously mapped to 8p23.1-p22 (KWE critical region) in South African families. Using targeted resequencing of the KWE critical region in five South African families and SNP array and whole-genome sequencing in two Norwegian families, we identified two overlapping tandem duplications of 7.67 kb (South Africans) and 15.93 kb (Norwegians). The duplications segregated with the disease and were located upstream of CTSB, a gene encoding cathepsin B, a cysteine protease involved in keratinocyte homeostasis. Included in the 2.62 kb overlapping region of these duplications is an enhancer element that is active in epidermal keratinocytes. The activity of this enhancer correlated with CTSB expression in normal differentiating keratinocytes and other cell lines, but not with FDFT1 or NEIL2 expression. Gene expression (qPCR) analysis and immunohistochemistry of the palmar epidermis demonstrated significantly increased expression of CTSB, as well as stronger staining of cathepsin B in the stratum granulosum of affected individuals than in that of control individuals. Analysis of higher-order chromatin structure data and RNA polymerase II ChIA-PET data from MCF-7 cells did not suggest remote effects of the enhancer. In conclusion, KWE in South African and Norwegian families is caused by tandem duplications in a non-coding genomic region containing an active enhancer element for CTSB, resulting in upregulation of this gene in affected individuals. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

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Vigani, Gianpiero; Rolli, Eleonora; Marasco, Ramona; Dell'Orto, Marta; Michoud, Grégoire; Soussi, Asma; Raddadi, Noura; Borin, Sara; Sorlini, Claudia; Zocchi, Graziano; Daffonchio, Daniele

2018-05-22

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H + -ATPase (V-ATPase) and H + -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H + -ATPase (V-ATPase) and H + -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Enhancement of inflammatory protein expression and nuclear factor Κb (NF-Κb) activity by trichostatin A (TSA) in OP9 preadipocytes.

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Sato, Taiki; Kotake, Daisuke; Hiratsuka, Masahiro; Hirasawa, Noriyasu

2013-01-01

The production of inflammatory proteins such as interleukin-6 (IL-6) by preadipocytes and mature adipocytes is closely associated with the impairment of systemic glucose homeostasis. However, precisely how the production is regulated and the roles of histone deacetylases (HDACs) remain largely unknown. The aim of this study was to establish whether HDAC inhibitors affect the expression of inflammatory proteins in pre/mature adipocytes, and, if so, to determine the mechanism involved. Trichostatin A (TSA), an HDAC inhibitor, enhanced lipopolysaccharide (LPS)-induced production of IL-6 in OP9 preadipocytes but not the mature adipocytes. Moreover, TSA also enhanced palmitic acid-induced IL-6 production and the expression of inflammatory genes induced by LPS in preadipocytes. Although TSA did not affect TLR4 mRNA expression or the activation of MAPKs, a reporter gene assay revealed that the LPS-induced increase in nuclear factor κB (NF-κB) activity was enhanced by TSA. Moreover, TSA increased the level of NF-κB p65 acetylation at lysine 310 and duration of its translocation into the nucleus, which leads to enhancement of NF-κB activity and subsequently expression of inflammatory genes. These findings shed new light on the regulatory roles of HDACs in preadipocytes in the production of inflammatory proteins.

Dual DNA methylation patterns in the CNS reveal developmentally poised chromatin and monoallelic expression of critical genes.

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Jinhui Wang

Full Text Available As a first step towards discovery of genes expressed from only one allele in the CNS, we used a tiling array assay for DNA sequences that are both methylated and unmethylated (the MAUD assay. We analyzed regulatory regions of the entire mouse brain transcriptome, and found that approximately 10% of the genes assayed showed dual DNA methylation patterns. They include a large subset of genes that display marks of both active and silent, i.e., poised, chromatin during development, consistent with a link between differential DNA methylation and lineage-specific differentiation within the CNS. Sixty-five of the MAUD hits and 57 other genes whose function is of relevance to CNS development and/or disorders were tested for allele-specific expression in F(1 hybrid clonal neural stem cell (NSC lines. Eight MAUD hits and one additional gene showed such expression. They include Lgi1, which causes a subtype of inherited epilepsy that displays autosomal dominance with incomplete penetrance; Gfra2, a receptor for glial cell line-derived neurotrophic factor GDNF that has been linked to kindling epilepsy; Unc5a, a netrin-1 receptor important in neurodevelopment; and Cspg4, a membrane chondroitin sulfate proteoglycan associated with malignant melanoma and astrocytoma in human. Three of the genes, Camk2a, Kcnc4, and Unc5a, show preferential expression of the same allele in all clonal NSC lines tested. The other six genes show a stochastic pattern of monoallelic expression in some NSC lines and bi-allelic expression in others. These results support the estimate that 1-2% of genes expressed in the CNS may be subject to allelic exclusion, and demonstrate that the group includes genes implicated in major disorders of the CNS as well as neurodevelopment.

Endoplasmic Reticulum Stress Sensor IRE1α Enhances IL-23 Expression by Human Dendritic Cells

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Saioa Márquez

2017-06-01

Full Text Available Human monocyte-derived dendritic cells (DCs exposed to pathogen-associated molecular patterns (PAMPs undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein β-dependent trans-activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs.

Composition and dosage of a multipartite enhancer cluster control developmental expression of Ihh (Indian hedgehog).

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Will, Anja J; Cova, Giulia; Osterwalder, Marco; Chan, Wing-Lee; Wittler, Lars; Brieske, Norbert; Heinrich, Verena; de Villartay, Jean-Pierre; Vingron, Martin; Klopocki, Eva; Visel, Axel; Lupiáñez, Darío G; Mundlos, Stefan

2017-10-01

Copy number variations (CNVs) often include noncoding sequences and putative enhancers, but how these rearrangements induce disease is poorly understood. Here we investigate CNVs involving the regulatory landscape of IHH (encoding Indian hedgehog), which cause multiple, highly localized phenotypes including craniosynostosis and synpolydactyly. We show through transgenic reporter and genome-editing studies in mice that Ihh is regulated by a constellation of at least nine enhancers with individual tissue specificities in the digit anlagen, growth plates, skull sutures and fingertips. Consecutive deletions, resulting in growth defects of the skull and long bones, showed that these enhancers function in an additive manner. Duplications, in contrast, caused not only dose-dependent upregulation but also misexpression of Ihh, leading to abnormal phalanges, fusion of sutures and syndactyly. Thus, precise spatiotemporal control of developmental gene expression is achieved by complex multipartite enhancer ensembles. Alterations in the composition of such clusters can result in gene misexpression and disease.

The enhancement of radiosensitivity by celecoxib, selective cyclooxygenase-2 inhibitor, on human cancer cells expressing differential levels of cyclooxygenase-2

International Nuclear Information System (INIS)

Pyo, Hong Ryull; Shin, You Keun; Kim, Hyun Seok; Seong, Jin Sil; Suh, Chang Ok; Kim, Gwi Eon

2003-01-01

To investigate the modulation of radiosensitivity by celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on cancer cells over- and under-expressing COX-2. A clonogenic radiation survival analysis was performed on A549 human lung and MCF-7 human breast cancer cell lines incubated in both 1 and 10% fetal bovine serum (FBS) containing media. The apoptosis in both cell lines was measured after treatment with radiation and/or celecoxib. Celecoxib enhanced the radiation sensitivity of the A549 cells in the medium containing the 10% FBS, with radiation enhancement ratios of 1.58 and 1.81 respectively, at surviving fractions of 0.1, with 30 μ M and 50 μ M celecoxib. This enhanced radiosensitivity disappeared in the medium containing the 1% FBS. Celecoxib did not change the radiation sensitivity of the MCF-7 cells in either media. The induction of apoptosis by celecoxib and radiation was not synergistic in either cell line. Celecoxib, a selective COX-2 inhibitor, preferentially enhanced the effect of radiation on COX-2 over-expressing cancer cells compared to the cells with a low expression, and this effect disappeared on incubation of the cells during drug treatment in the medium with suboptimal serum concentration. Apoptosis did not appear to be the underlying mechanism of this radiation enhancement effect due to celecoxib on the A549 cells. These findings suggest radiosensitization by a selective COX-2 inhibitor is COX-2 dependent

Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract

DEFF Research Database (Denmark)

Connell, Hugh; Agace, William; Klemm, Per

1996-01-01

of Escherichia coli for the urinary tract by promoting bacterial persistence and enhancing the inflammatory responce to infection. In a clinical study, we observed that disease severity was greater in children infected with E. coli O1:K1:H7 isolates expressing type 1 fimbriae than in those infected with type 1...... negative isolates of the same serotype. The E. coli O1:K1:H7 isolates had the same electrophoretic type, were hemolysin-negative, expressed P fimbriae, and carried the fim DNA sequences. When tested in a mouse urinary tract infection model, the type 1-positive E. coli O1:K1:H7 isolates survived inhigher...... urinary tract infection model. E. coli CN1016 reconstituted with type 1 fimbriae had restored virulence similar to that of the wild-type parent strain. These results show that type 1 fimbriae in the genetic background of a uropathogenic strain contribute to the pathogenesis of E. coli in the urinary tract....

Dynamic Displays Enhance the Ability to Discriminate Genuine and Posed Facial Expressions of Emotion

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Namba, Shushi; Kabir, Russell S.; Miyatani, Makoto; Nakao, Takashi

2018-01-01

Accurately gauging the emotional experience of another person is important for navigating interpersonal interactions. This study investigated whether perceivers are capable of distinguishing between unintentionally expressed (genuine) and intentionally manipulated (posed) facial expressions attributed to four major emotions: amusement, disgust, sadness, and surprise. Sensitivity to this discrimination was explored by comparing unstaged dynamic and static facial stimuli and analyzing the results with signal detection theory. Participants indicated whether facial stimuli presented on a screen depicted a person showing a given emotion and whether that person was feeling a given emotion. The results showed that genuine displays were evaluated more as felt expressions than posed displays for all target emotions presented. In addition, sensitivity to the perception of emotional experience, or discriminability, was enhanced in dynamic facial displays, but was less pronounced in the case of static displays. This finding indicates that dynamic information in facial displays contributes to the ability to accurately infer the emotional experiences of another person. PMID:29896135

Enhancement of extracellular expression of Bacillus naganoensis pullulanase from recombinant Bacillus subtilis: Effects of promoter and host.

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Song, Wan; Nie, Yao; Mu, Xiao Qing; Xu, Yan

2016-08-01

Pullulanase plays an important role in industrial applications of starch processing. However, extracellular production of pullulanase from recombinant Bacillus subtilis is yet limited due to the issues on regulatory elements of B. subtilis expression system. In this study, the gene encoding B. naganoensis pullulanase (PUL) was expressed in B. subtilis WB800 under the promoter PHpaII in the shuttle vector pMA0911. The extracellular activity of expressed pullulanase was 3.9 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-PHpaII-pul. To further enhance the yield of PUL, the promoter PHpaII in pMA0911 was replaced by a stronger constitutive promoter P43. Then the activity was increased to 8.7 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-P43-pul. Effect of host on pullulanase expression was further investigated by comparison between B. subtilis WB600 and B. subtilis WB800. In addition to the available B. subtilis WB800 recombinants, the constructed plasmids pMA0911-PHpaII-pul and pMA0911-P43-pul were transformed into B. subtilis WB600, respectively. Consequently, the extracellular production of PUL was significantly enhanced by B. subtilis WB600/pMA0911-P43-pul, resulting in the extracellular pullulanase activity of 24.5 U ml(-1). Therefore, promoter and host had an impact on pullulanase expression and their optimization would be useful to improve heterologous protein expression in B. subtilis. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

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Yu-Guo Yuan

2014-01-01

Full Text Available To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC containing a hybrid goat β-lactoglobulin (BLG promoter/cytomegalovirus (CMV enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT. Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2% from the non-transfected line and five recipients delivered six kids (1.8% from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P<0.05. Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

Tricyclic antidepressant amitriptyline indirectly increases the proliferation of adult dentate gyrus-derived neural precursors: an involvement of astrocytes.

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Shuken Boku

Full Text Available Antidepressants increase the proliferation of neural precursors in adult dentate gyrus (DG, which is considered to be involved in the therapeutic action of antidepressants. However, the mechanism underlying it remains unclear. By using cultured adult rat DG-derived neural precursors (ADP, we have already shown that antidepressants have no direct effects on ADP. Therefore, antidepressants may increase the proliferation of neural precursors in adult DG via unknown indirect mechanism. We have also shown that amitriptyline (AMI, a tricyclic antidepressant, induces the expressions of GDNF, BDNF, FGF2 and VEGF, common neurogenic factors, in primary cultured astrocytes (PCA. These suggest that AMI-induced factors in astrocytes may increase the proliferation of neural precursors in adult DG. To test this hypothesis, we examined the effects of AMI-induced factors and conditioned medium (CM from PCA treated with AMI on ADP proliferation. The effects of CM and factors on ADP proliferation were examined with BrdU immunocytochemistry. AMI had no effect on ADP proliferation, but AMI-treated CM increased it. The receptors of GDNF, BDNF and FGF2, but not VEGF, were expressed in ADP. FGF2 significantly increased ADP proliferation, but not BDNF and GDNF. In addition, both of a specific inhibitor of FGF receptors and anti-FGF2 antibody significantly counteracted the increasing effect of CM on ADP proliferation. In addition, FGF2 in brain is mainly derived from astrocytes that are key components of the neurogenic niches in adult DG. These suggest that AMI may increase ADP proliferation indirectly via PCA and that FGF2 may a potential candidate to mediate such an indirect effect of AMI on ADP proliferation via astrocytes.

DEVELOPING VISUAL NOVEL GAME WITH SPEECH-RECOGNITION INTERACTIVITY TO ENHANCE STUDENTS’ MASTERY ON ENGLISH EXPRESSIONS

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Elizabeth Anggraeni Amalo

2017-11-01

Full Text Available The teaching of English-expressions has always been done through conversation samples in form of written texts, audio recordings, and videos. In the meantime, the development of computer-aided learning technology has made autonomous language learning possible. Game, as one of computer-aided learning technology products, can serve as a medium to provide educational contents like that of language teaching and learning. Visual Novel is considered as a conversational game that is suitable to be combined with English-expressions material. Unlike the other click-based interaction Visual Novel Games, the visual novel game in this research implements speech recognition as the interaction trigger. Hence, this paper aims at elaborating how visual novel games are utilized to deliver English-expressions with speech recognition command for the interaction. This research used Research and Development (R&D method with Experimental design through control and experimental groups to measure its effectiveness in enhancing students’ English-expressions mastery. ANOVA was utilized to prove the significant differences between the control and experimental groups. It is expected that the result of this development and experiment can devote benefits to the English teaching and learning, especially on English-expressions.

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium.

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Peng, Lin; Yu, Xiao; Li, Chengyuan; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

2016-04-01

Signal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

Optimization of recombinant bacteria expressing dsRNA to enhance insecticidal activity against a lepidopteran insect, Spodoptera exigua.

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Mohammad Vatanparast

Full Text Available Double-stranded RNA (dsRNA has been applied to control insect pests due to its induction of RNA interference (RNAi of a specific target gene expression. However, developing dsRNA-based insecticidal agent has been a great challenge especially against lepidopteran insect pests due to variations in RNAi efficiency. The objective of this study was to screen genes of chymotrypsins (SeCHYs essential for the survival of the beet armyworm, Spodoptera exigua, to construct insecticidal dsRNA. In addition, an optimal oral delivery method was developed using recombinant bacteria. At least 7 SeCHY genes were predicted from S. exigua transcriptomes. Subsequent analyses indicated that SeCHY2 was widely expressed in different developmental stages and larval tissues by RT-PCR and its expression knockdown by RNAi caused high mortality along with immunosuppression. However, a large amount of dsRNA was required to efficiently kill late instars of S. exigua because of high RNase activity in their midgut lumen. To minimize dsRNA degradation, bacterial expression and formulation of dsRNA were performed in HT115 Escherichia coli using L4440 expression vector. dsRNA (300 bp specific to SeCHY2 overexpressed in E. coli was toxic to S. exigua larvae after oral administration. To enhance dsRNA release from E. coli, bacterial cells were sonicated before oral administration. RNAi efficiency of sonicated bacteria was significantly increased, causing higher larval mortality at oral administration. Moreover, targeting young larvae possessing weak RNase activity in the midgut lumen significantly enhanced RNAi efficiency and subsequent insecticidal activity against S. exigua.

The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway.

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Wang, Songbo; Wang, Guoqing; Zhang, Mengyuan; Zhuang, Lu; Wan, Xiaojuan; Xu, Jingren; Wang, Lina; Zhu, Xiaotong; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

2016-11-15

It has been implicated that IGF-1 secretion can be regulated by dietary protein. However, whether the dipeptides, one of digested products of dietary protein, have influence on IGF-1 secretion remain largely unknown. Our study aimed to investigate the effects of the dipeptide Pro-Asp on IGF-1 secretion and expression in hepatocytes and to explore the possible underlying mechanisms. Our findings demonstrated that Pro-Asp promoted the secretion and gene expression of IGF-1 in HepG2 cells and primary porcine hepatocytes. Meanwhile, Pro-Asp activated the ERK and Akt signaling pathways, downstream of IGF-1. In addition, Pro-Asp enhanced GH-mediated JAK2/STAT5 signaling pathway, while inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Asp on IGF-1 secretion and expression. Moreover, acute injection of Pro-Asp stimulated IGF-1 expression and activated JAK2/STAT5 signaling pathway in mice liver. Together, these results suggested that the dipeptide Pro-Asp promoted IGF-1 secretion and expression in hepatocytes by enhancing GH-mediated JAK2/STAT5 signaling pathway. Copyright © 2016. Published by Elsevier Ireland Ltd.

Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression in Jurkat Cells.

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Pan, Yao; Wei, Xuetao; Hao, Weidong

2015-08-28

Trichloroethylene (TCE) is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells.

Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression in Jurkat Cells

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Yao Pan

2015-08-01

Full Text Available Trichloroethylene (TCE is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA and dichloroacetic acid (DCA, on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells.

Elevated expression of proto-oncogenes accompany enhanced induction of heat-shock genes after exposure of rat embryos in utero to ionizing irradiation

International Nuclear Information System (INIS)

Higo, H.; Lee, J.Y.; Satow, Y.; Higo, K.

1989-01-01

We have recently found that the effects of exposing rat embryos in utero to teratogens capable of producing cardiac anomalies were expressed later as enhanced induction of heat-shock proteins (hsp70 family) when embryonic hearts were cultured in vitro. However, it remained to be determined whether heat-shock proteins are induced in vivo after exposure to teratogens. The heat-shock response in some mammalian systems is known to be accompanied by elevated expression of proto-oncogenes. Using gene-specific DNA probes, we examined the levels of the expression (transcription) of heat-shock protein genes and two nuclear proto-oncogenes, c-fos and c-myc, in the embryos removed from irradiated pregnant mother rats 4 or 5 days after the irradiation. We found that the levels of expression in vivo of the hsp70 and c-myc genes in the irradiated embryos increased by approximately twofold as compared with those in the control. The expression in vivo of the c-fos gene was not detected in either the irradiated or non-irradiated embryos. After 0.5-hr incubation in vitro of the embryos, however, the expression of the c-fos gene in the irradiated embryos was highly enhanced whereas the control showed no changes. Although the exact functions of these gene products still remain obscure, the enhanced expression of hsp70 gene(s) and the nuclear proto-oncogenes observed in the present study may reflect repair of intracellular damages and/or regeneration of tissue by compensatory cell proliferation, processes that may disturb the normal program of organogenesis

Overexpression of LOV KELCH protein 2 confers dehydration tolerance and is associated with enhanced expression of dehydration-inducible genes in Arabidopsis thaliana.

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Miyazaki, Yuji; Abe, Hiroshi; Takase, Tomoyuki; Kobayashi, Masatomo; Kiyosue, Tomohiro

2015-05-01

The overexpression of LKP2 confers dehydration tolerance in Arabidopsis thaliana ; this is likely due to enhanced expression of dehydration-inducible genes and reduced stomatal opening. LOV KELCH protein 2 (LKP2) modulates the circadian rhythm and flowering time in plants. In this study, we observed that LKP2 overexpression enhanced dehydration tolerance in Arabidopsis. Microarray analysis demonstrated that expression of water deprivation-responsive genes was higher in the absence of dehydration stress in transgenic Arabidopsis plants expressing green fluorescent protein-tagged LKP2 (GFP-LKP2) than in control transgenic plants expressing GFP. After dehydration followed by rehydration, GFP-LKP2 plants developed more leaves and roots and exhibited higher survival rates than control plants. In the absence of dehydration stress, four dehydration-inducible genes, namely DREB1A, DREB1B, DREB1C, and RD29A, were expressed in GFP-LKP2 plants, whereas they were not expressed or were expressed at low levels in control plants. Under dehydration stress, the expression of DREB2B and RD29A peaked faster in the GFP-LKP2 plants than in control plants. The stomatal aperture of GFP-LKP2 plants was smaller than that of control plants. These results suggest that the dehydration tolerance of GFP-LKP2 plants is caused by upregulation of DREB1A-C/CBF1-3 and their downstream targets; restricted stomatal opening in the absence of dehydration stress also appears to contribute to the phenotype. The rapid and high expression of DREB2B and its downstream target genes also likely accounts for some features of the GFP-LKP2 phenotype. Our results suggest that LKP2 can be used for biotechnological applications not only to adjust the flowering time control but also to enhance dehydration tolerance.

Retinal pigment epithelial cells secrete neurotrophic factors and synthesize dopamine: possible contribution to therapeutic effects of RPE cell transplantation in Parkinson's disease

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Gu Qing

2009-06-01

Full Text Available Abstract Background New strategies for the treatment of Parkinson's disease (PD are shifted from dopamine (DA replacement to regeneration or restoration of the nigro-striatal system. A cell therapy using human retinal pigment epithelial (RPE cells as substitution for degenerated dopaminergic (DAergic neurons has been developed and showed promising prospect in clinical treatment of PD, but the exact mechanism underlying this therapy is not fully elucidated. In the present study, we investigated whether the beneficial effects of this therapy are related to the trophic properties of RPE cells and their ability to synthesize DA. Methods We evaluated the protective effects of conditioned medium (CM from cultured RPE cells on the DAergic cells against 6-hydroxydopamine (6-OHDA- and rotenone-induced neurotoxicity and determined the levels of glial cell derived neurotrophic factor (GDNF and brain derived neurotrophic factor (BDNF released by RPE cells. We also measured the DA synthesis and release. Finally we transplanted microcarriers-RPE cells into 6-OHDA lesioned rats and observed the improvement in apomorphine-induced rotations (AIR. Results We report here: (1 CM from RPE cells can secret trophic factors GDNF and BDNF, and protect DAergic neurons against the 6-OHDA- and rotenone-induced cell injury; (2 cultured RPE cells express L-dopa decarboxylase (DDC and synthesize DA; (3 RPE cells attached to microcarriers can survive in the host striatum and improve the AIR in 6-OHDA-lesioned animal model of PD; (4 GDNF and BDNF levels are found significantly higher in the RPE cell-grafted tissues. Conclusion These findings indicate the RPE cells have the ability to secret GDNF and BDNF, and synthesize DA, which probably contribute to the therapeutic effects of RPE cell transplantation in PD.

Transplastomic expression of bacterial L-aspartate-alpha-decarboxylase enhances photosynthesis and biomass production in response to high temperature stress.

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Fouad, W M; Altpeter, F

2009-10-01

Metabolic engineering for beta-alanine over-production in plants is expected to enhance environmental stress tolerance. The Escherichia coli L-aspartate-alpha-decarboxylase (AspDC) encoded by the panD gene, catalyzes the decarboxylation of L-aspartate to generate beta-alanine and carbon dioxide. The constitutive E. coli panD expression cassette was co-introduced with the constitutive, selectable aadA expression cassette into the chloroplast genome of tobacco via biolistic gene transfer and homologous recombination. Site specific integration of the E. coli panD expression cassette into the chloroplast genome and generation of homotransplastomic plants were confirmed by PCR and Southern blot analysis, respectively, following plant regeneration and germination of seedlings on selective media. PanD expression was verified by assays based on transcript detection and in vitro enzyme activity. The AspDC activities in transplastomic plants expressing panD were drastically increased by high-temperature stress. beta-Alanine accumulated in transplastomic plants at levels four times higher than in wildtype plants. Analysis of chlorophyll fluorescence on plants subjected to severe heat stress at 45 degrees C under light verified that photosystem II (PSII) in transgenic plants had higher thermotolerance than in wildtype plants. The CO(2) assimilation of transplastomic plants expressing panD was more tolerant to high temperature stress than that of wildtype plants, resulting in the production of 30-40% more above ground biomass than wildtype control. The results presented indicate that chloroplast engineering of the beta-alanine pathway by over-expression of the E. coli panD enhances thermotolerance of photosynthesis and biomass production following high temperature stress.

FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

International Nuclear Information System (INIS)

Eyre, Nicholas S.; Cleland, Leslie G.; Mayrhofer, Graham

2008-01-01

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. We propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells

Comparative study of the distribution of the alpha-subunits of voltage-gated sodium channels in normal and axotomized rat dorsal root ganglion neurons.

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Fukuoka, Tetsuo; Kobayashi, Kimiko; Yamanaka, Hiroki; Obata, Koichi; Dai, Yi; Noguchi, Koichi

2008-09-10

We compared the distribution of the alpha-subunit mRNAs of voltage-gated sodium channels Nav1.1-1.3 and Nav1.6-1.9 and a related channel, Nax, in histochemically identified neuronal subpopulations of the rat dorsal root ganglia (DRG). In the naïve DRG, the expression of Nav1.1 and Nav1.6 was restricted to A-fiber neurons, and they were preferentially expressed by TrkC neurons, suggesting that proprioceptive neurons possess these channels. Nav1.7, -1.8, and -1.9 mRNAs were more abundant in C-fiber neurons compared with A-fiber ones. Nax was evenly expressed in both populations. Although Nav1.8 and -1.9 were preferentially expressed by TrkA neurons, other alpha-subunits were expressed independently of TrkA expression. Actually, all IB4(+) neurons expressed both Nav1.8 and -1.9, and relatively limited subpopulations of IB4(+) neurons (3% and 12%, respectively) expressed Nav1.1 and/or Nav1.6. These findings provide useful information in interpreting the electrophysiological characteristics of some neuronal subpopulations of naïve DRG. After L5 spinal nerve ligation, Nav1.3 mRNA was up-regulated mainly in A-fiber neurons in the ipsilateral L5 DRG. Although previous studies demonstrated that nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) reversed this up-regulation, the Nav1.3 induction was independent of either TrkA or GFRalpha1 expression, suggesting that the induction of Nav1.3 may be one of the common responses of axotomized DRG neurons without a direct relationship to NGF/GDNF supply. (c) 2008 Wiley-Liss, Inc.

Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability.

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Ruiz, Oscar N; Alvarez, Derry; Torres, Cesar; Roman, Laura; Daniell, Henry

2011-06-01

Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy. Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183,000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 μm mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioneins in plants for mercury phytoremediation. Chloroplast genetic engineering approach is useful to express metal-scavenging proteins for phytoremediation. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Cloning of radiation-induced new gene RS1 expressed in mouse intestinal epithelium by enhanced RACE

International Nuclear Information System (INIS)

Wang Fengchao; Wang Junping; Su Yongping; Gao Jinsheng; Lou Shufen; Liu Xiaohong; Ren Jiong; Zhang Bo

2003-01-01

Objective: To obtain full-length cDNA of radiation-induced new gene RS1 expressed in mouse intestinal epithelium. Methods: The tissue expression profile of RS1 was analyzed by semi-quantitative RT-PCR to find the target tissue which highly expresses RS1. The total RNA extracted from the corresponding tissue was taken as the template for reverse-transcription. Enhanced RACE PCR was used to clone the full-length cDNA of RS1, including enrichment of the target gene through biotin-labeled probe for magnetic bead purification and nested PCR. Results: About a 2 kb long 3' end was successfully cloned and cloning of the 5' end proceeded well. Conclusion: The result is consistent with our experiment design. The set of combined techniques has been identified with the cloning of full-length cDNA from EST sequence especially when the optimal gene-specific primers are not available or the expression level of target gene is low

Correlation of emmprin expression in vascular endothelial cells with blood-brain-barrier function: a study using magnetic resonance imaging enhanced by Gd-DTPA and immunohistochemistry in brain tumors.

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Sameshima, Tetsuro; Nabeshima, Kazuki; Toole, Bryan P; Inoue, Teruhiko; Yokogami, Kiyotaka; Nakano, Shinichi; Ohi, Takekazu; Wakisaka, Shinichiro

2003-06-01

In a previous study, we demonstrated that the expression levels in tumor cells of emmprin (CD147) correlated with the grade of astrocytic tumors. Also, we found that emmprin was expressed in vascular endothelial cells of the non-neoplastic brain and hypothesized that emmprin expression could be associated with normal blood-brain-barrier (BBB) function of vascular endothelial cells. In this study, this possibility was examined in non-neoplastic brain, glioma and metastatic carcinoma tissues by comparing emmprin immunohistochemistry with gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) enhancement of magnetic resonance imaging (MRI), which is a clinical indicator of the BBB function. This study included 10 cases of non-neoplastic brain tissues, 7 of metastatic carcinoma, 7 of diffuse astrocytoma, 4 of anaplastic astrocytoma and 13 of glioblastoma multiforme. In all the cases, MRI with administration of Gd-DTPA was performed. The lesions were resected using the microdissection method with the help of ultrasonography and a neuronavigator. The tissues from Gd-DTPA-enhanced or non-enhanced areas were processed into frozen sections and subjected to immunohistochemistry with anti-emmprin antibody. The expression of emmprin in brain vascular endothelial cells inversely correlated with Gd-DTPA-enhancement of MRI: emmprin was positive in tissues not enhanced by Gd-DTPA and was negative in DTPA-enhanced tissues. Since BBB function presumably remains unimpaired in regions in which MR images are not Gd-DTPA-enhanced, emmprin expression appears to be associated with unimpaired BBB function. This is the first report to demonstrate a possible correlation between emmprin expression and BBB function in humans.

TWEAK enhances E-selectin and ICAM-1 expression, and may contribute to the development of cutaneous vasculitis.

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Tao Chen

Full Text Available Our previous work indicated that TWEAK is associated with various types of cutaneous vasculitis (CV. Herein, we investigate the effects of TWEAK on vascular injury and adhesion molecule expression in CV mice. We showed that TWEAK priming in mice induced a local CV. Furthermore, TWEAK priming also increased the extravasation of FITC-BSA, myeloperoxidase activity and the expression of E-selectin and ICAM-1. Conversely, TWEAK blockade ameliorated the LPS-induced vascular damage, leukocyte infiltrates and adhesion molecules expression in LPS-induced CV. In addition, TWEAK treatment of HDMECs up-regulated E-selectin and ICAM-1 expression at both mRNA and protein levels. TWEAK also enhanced the adhesion of PMNs to HDMECs. Finally, western blot data revealed that TWEAK can induce phosphorylation of p38, JNK and ERK in HDMECs. These data suggest that TWEAK acted as an inducer of E-selectin and ICAM-1 expression in CV mice and HDMECs, may contribute to the development of CV.

Largazole, a class I histone deacetylase inhibitor, enhances TNF-α-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts

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Ahmed, Salahuddin, E-mail: Salah.Ahmed@utoledo.edu [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Riegsecker, Sharayah; Beamer, Maria; Rahman, Ayesha; Bellini, Joseph V. [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Bhansali, Pravin; Tillekeratne, L.M. Viranga [Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States)

2013-07-15

In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1–5 μM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5–5 μM) inhibited the constitutive expression of HDAC1 (0–30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ∼ 220% with a concomitant decrease in HDAC5 [30–58%] expression in RA synovial fibroblasts. SAHA (5 μM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α + LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA. - Highlights: • Largazole enhances TNF-α-induced ICAM-1 and VCAM-1. • Largazole upregulates class II HDAC (HDAC6) in RA synovial fibroblasts. • Largazole also induces the expression of phospho-p38

Inhibition of Super-Enhancer Activity in Autoinflammatory Site-Derived T Cells Reduces Disease-Associated Gene Expression.

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Peeters, Janneke G C; Vervoort, Stephin J; Tan, Sander C; Mijnheer, Gerdien; de Roock, Sytze; Vastert, Sebastiaan J; Nieuwenhuis, Edward E S; van Wijk, Femke; Prakken, Berent J; Creyghton, Menno P; Coffer, Paul J; Mokry, Michal; van Loosdregt, Jorg

2015-09-29

The underlying molecular mechanisms for many autoimmune diseases are poorly understood. Juvenile idiopathic arthritis (JIA) is an exceptionally well-suited model for studying autoimmune diseases due to its early onset and the possibility to analyze cells derived from the site of inflammation. Epigenetic profiling, utilizing primary JIA patient-derived cells, can contribute to the understanding of autoimmune diseases. With H3K27ac chromatin immunoprecipitation, we identified a disease-specific, inflammation-associated, typical enhancer and super-enhancer signature in JIA patient synovial-fluid-derived CD4(+) memory/effector T cells. RNA sequencing of autoinflammatory site-derived patient T cells revealed that BET inhibition, utilizing JQ1, inhibited immune-related super-enhancers and preferentially reduced disease-associated gene expression, including cytokine-related processes. Altogether, these results demonstrate the potential use of enhancer profiling to identify disease mediators and provide evidence for BET inhibition as a possible therapeutic approach for the treatment of autoimmune diseases. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Inhibition of Super-Enhancer Activity in Autoinflammatory Site-Derived T Cells Reduces Disease-Associated Gene Expression

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Janneke G.C. Peeters

2015-09-01

Full Text Available The underlying molecular mechanisms for many autoimmune diseases are poorly understood. Juvenile idiopathic arthritis (JIA is an exceptionally well-suited model for studying autoimmune diseases due to its early onset and the possibility to analyze cells derived from the site of inflammation. Epigenetic profiling, utilizing primary JIA patient-derived cells, can contribute to the understanding of autoimmune diseases. With H3K27ac chromatin immunoprecipitation, we identified a disease-specific, inflammation-associated, typical enhancer and super-enhancer signature in JIA patient synovial-fluid-derived CD4+ memory/effector T cells. RNA sequencing of autoinflammatory site-derived patient T cells revealed that BET inhibition, utilizing JQ1, inhibited immune-related super-enhancers and preferentially reduced disease-associated gene expression, including cytokine-related processes. Altogether, these results demonstrate the potential use of enhancer profiling to identify disease mediators and provide evidence for BET inhibition as a possible therapeutic approach for the treatment of autoimmune diseases.

Lymphocytes from wasted mice express enhanced spontaneous and {gamma}-ray-induced apoptosis

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Woloschak, G.E. [Argonne National Lab., IL (United States)]|[Loyola Univ. Medical Center, Maywood, IL (United States); Chang-Liu, Chin-Mei [Argonne National Lab., IL (United States); Chung, Jen; Libertin, C.R. [Loyola Univ. Medical Center, Maywood, IL (United States)

1993-09-01

Mice bearing the autosomal recessive mutation wasted (wst/wst) display a disease pattern including faulty repair of DNA damage in lymphocytes after radiation exposure, neurologic abnormalities, and immunodeficiency. Many of the features of this mouse model have suggested a premature or increased spontaneous frequency of apoptosis in thymocytes; past work has shown an inability to establish cultured T cell lines, an abnormally high death rate of stimulated T cells in culture, and an increased sensitivity of T cells to the killing effects of ionizing radiations in wst/wst mice relative to controls. The experiments reported here were designed to examine splenic and thymic lymphocytes from wasted and control mice for signs of early apoptosis. Our results revealed enhanced expression of Rp-8 mRNA (associated with apoptosis) in thymic lymphocytes and reduced expression in splenic lymphocytes of wst/wst mice relative to controls; expression of Rp-2 and Td-30 mRNA (induced during apoptosis) were not detectable in spleen or thymus. Higher spontaneous DNA fragmentation was observed in wasted mice than in controls; however, {gamma}-ray-induced DNA fragmentation peaked at a lower dose and occurred to a greater extent in wasted mice relative to controls. These results provide evidence for high spontaneous and {gamma}-ray-induced apoptosis in T cells of wasted mice as a mechanism underlying the observed lymphocyte and DNA repair abnormalities.

Nimotuzumab enhances temozolomide?induced growth suppression of glioma cells expressing mutant EGFR in vivo

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Nitta, Yusuke; Shimizu, Saki; Shishido?Hara, Yukiko; Suzuki, Kaori; Shiokawa, Yoshiaki; Nagane, Motoo

2016-01-01

Abstract A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti?EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild?type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and pho...

Let the Avatar Brighten Your Smile: Effects of Enhancing Facial Expressions in Virtual Environments.

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Soo Youn Oh

Full Text Available Previous studies demonstrated the positive effects of smiling on interpersonal outcomes. The present research examined if enhancing one's smile in a virtual environment could lead to a more positive communication experience. In the current study, participants' facial expressions were tracked and mapped on a digital avatar during a real-time dyadic conversation. The avatar's smile was rendered such that it was either a slightly enhanced version or a veridical version of the participant's actual smile. Linguistic analyses using the Linguistic Inquiry Word Count (LIWC revealed that participants who communicated with each other via avatars that exhibited enhanced smiles used more positive words to describe their interaction experience compared to those who communicated via avatars that displayed smiling behavior reflecting the participants' actual smiles. In addition, self-report measures showed that participants in the 'enhanced smile' condition felt more positive affect after the conversation and experienced stronger social presence compared to the 'normal smile' condition. These results are particularly striking when considering the fact that most participants (>90% were unable to detect the smiling manipulation. This is the first study to demonstrate the positive effects of transforming unacquainted individuals' actual smiling behavior during a real-time avatar-networked conversation.

Expression of an Arabidopsis molybdenum cofactor sulphurase gene in soybean enhances drought tolerance and increases yield under field conditions.

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Li, Yajun; Zhang, Jiachang; Zhang, Juan; Hao, Ling; Hua, Jinping; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

2013-08-01

LOS5/ABA3 gene encoding molybdenum cofactor sulphurase is involved in aldehyde oxidase (AO) activity in Arabidopsis, which indirectly regulates ABA biosynthesis and increased stress tolerance. Here, we used a constitutive super promoter to drive LOS5/ABA3 overexpression in soybean (Glycine max L.) to enhance drought tolerance in growth chamber and field conditions. Expression of LOS5/ABA3 was up-regulated by drought stress, which led to increasing AO activity and then a notable increase in ABA accumulation. Transgenic soybean under drought stress had reduced water loss by decreased stomatal aperture size and transpiration rate, which alleviated leaf wilting and maintained higher relative water content. Exposed to drought stress, transgenic soybean exhibited reduced cell membrane damage by reducing electrolyte leakage and production of malondialdehyde and promoting proline accumulation and antioxidant enzyme activities. Also, overexpression of LOS5/ABA3 enhanced expression of stress-up-regulated genes. Furthermore, the seed yield of transgenic plants is at least 21% higher than that of wide-type plants under drought stress conditions in the field. These data suggest that overexpression of LOS5/ABA3 could improve drought tolerance in transgenic soybean via enhanced ABA accumulation, which could activate expression of stress-up-regulated genes and cause a series of physiological and biochemical resistant responses. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Enhancing cytochrome P450-mediated conversions in P. pastoris through RAD52 over-expression and optimizing the cultivation conditions.

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Wriessnegger, Tamara; Moser, Sandra; Emmerstorfer-Augustin, Anita; Leitner, Erich; Müller, Monika; Kaluzna, Iwona; Schürmann, Martin; Mink, Daniel; Pichler, Harald

2016-04-01

Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhanced tolerance and remediation of anthracene by transgenic tobacco plants expressing a fungal glutathione transferase gene

International Nuclear Information System (INIS)

Dixit, Prachy; Mukherjee, Prasun K.; Sherkhane, Pramod D.; Kale, Sharad P.; Eapen, Susan

2011-01-01

Highlights: → Transgenic plants expressing a TvGST gene were tested for tolerance, uptake and degradation of anthracene. → Transgenic plants were more tolerant to anthracene and take up more anthracene from soil and solutions compared to control plants. → Using in vitro T 1 seedlings, we showed that anthracene-a three fused benzene ring compound was phytodegraded to naphthalene derivatives, having two benzene rings. → This is the first time that a transgenic plant was shown to have the potential to phytodegrade anthracene. - Abstract: Plants can be used for remediation of polyaromatic hydrocarbons, which are known to be a major concern for human health. Metabolism of xenobiotic compounds in plants occurs in three phases and glutathione transferases (GST) mediate phase II of xenobiotic transformation. Plants, although have GSTs, they are not very efficient for degradation of exogenous recalcitrant xenobiotics including polyaromatic hydrocarbons. Hence, heterologous expression of efficient GSTs in plants may improve their remediation and degradation potential of xenobiotics. In the present study, we investigated the potential of transgenic tobacco plants expressing a Trichoderma virens GST for tolerance, remediation and degradation of anthracene-a recalcitrant polyaromatic hydrocarbon. Transgenic plants with fungal GST showed enhanced tolerance to anthracene compared to control plants. Remediation of 14 C uniformly labeled anthracene from solutions and soil by transgenic tobacco plants was higher compared to wild-type plants. Transgenic plants (T 0 and T 1 ) degraded anthracene to naphthalene derivatives, while no such degradation was observed in wild-type plants. The present work has shown that in planta expression of a fungal GST in tobacco imparted enhanced tolerance as well as higher remediation potential of anthracene compared to wild-type plants.

CCAAT/Enhancer Binding Protein β Regulates Expression of Indian Hedgehog during Chondrocytes Differentiation

Science.gov (United States)

Ushijima, Takahiro; Okazaki, Ken; Tsushima, Hidetoshi; Ishihara, Kohei; Doi, Toshio; Iwamoto, Yukihide

2014-01-01

Background CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh) also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2) was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation. Methodology/Results Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between −214 and −210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP) assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression. Conclusions C

CCAAT/enhancer binding protein β regulates expression of Indian hedgehog during chondrocytes differentiation.

Directory of Open Access Journals (Sweden)

Takahiro Ushijima

Full Text Available CCAAT/enhancer binding protein β (C/EBPβ is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2 was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation.Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between -214 and -210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA and a chromatin immunoprecipitation (ChIP assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression.C/EBPβ and RUNX2 cooperatively stimulate

Two estrogen response element sequences near the PCNA gene are not responsible for its estrogen-enhanced expression in MCF7 cells.

Science.gov (United States)

Wang, Cheng; Yu, Jie; Kallen, Caleb B

2008-01-01

The proliferating cell nuclear antigen (PCNA) is an essential component of DNA replication, cell cycle regulation, and epigenetic inheritance. High expression of PCNA is associated with poor prognosis in patients with breast cancer. The 5'-region of the PCNA gene contains two computationally-detected estrogen response element (ERE) sequences, one of which is evolutionarily conserved. Both of these sequences are of undocumented cis-regulatory function. We recently demonstrated that estradiol (E2) enhances PCNA mRNA expression in MCF7 breast cancer cells. MCF7 cells proliferate in response to E2. Here, we demonstrate that E2 rapidly enhanced PCNA mRNA and protein expression in a process that requires ERalpha as well as de novo protein synthesis. One of the two upstream ERE sequences was specifically bound by ERalpha-containing protein complexes, in vitro, in gel shift analysis. Yet, each ERE sequence, when cloned as a single copy, or when engineered as two tandem copies of the ERE-containing sequence, was not capable of activating a luciferase reporter construct in response to E2. In MCF7 cells, neither ERE-containing genomic region demonstrated E2-dependent recruitment of ERalpha by sensitive ChIP-PCR assays. We conclude that E2 enhances PCNA gene expression by an indirect process and that computational detection of EREs, even when evolutionarily conserved and when near E2-responsive genes, requires biochemical validation.

Hypoxia targeted bifunctional suicide gene expression enhances radiotherapy in vitro and in vivo

International Nuclear Information System (INIS)

Sun, Xiaorong; Xing, Ligang; Deng, Xuelong; Hsiao, Hung Tsung; Manami, Akiko; Koutcher, Jason A.; Clifton Ling, C.; Li, Gloria C.

2012-01-01

Purpose: To investigate whether hypoxia targeted bifunctional suicide gene expression-cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with 5-FC treatments can enhance radiotherapy. Materials and methods: Stable transfectants of R3327-AT cells were established which express a triple-fusion-gene: CD, UPRT and monomoric DsRed (mDsRed) controlled by a hypoxia inducible promoter. Hypoxia-induced expression/function of CDUPRTmDsRed was verified by western blot, flow cytometry, fluorescent microscopy, and cytotoxicity assay of 5-FU and 5-FC. Tumor-bearing mice were treated with 5-FC and local radiation. Tumor volume was monitored and compared with those treated with 5-FC or radiation alone. In addition, the CDUPRTmDsRed distribution in hypoxic regions of tumor sections was visualized with fluorescent microscopy. Results: Hypoxic induction of CDUPRTmDsRed protein correlated with increased sensitivity to 5-FC and 5-FU. Significant radiosensitization effects were detected after 5-FC treatments under hypoxic conditions. In the tumor xenografts, the distribution of CDUPRTmDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC to mice in combination with local irradiation resulted in significant tumor regression, as in comparison with 5-FC or radiation treatments alone. Conclusions: Our data suggest that the hypoxia-inducible CDUPRT/5-FC gene therapy strategy has the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone.

Science.gov (United States)

Gonen, Nitzan; Quinn, Alexander; O'Neill, Helen C; Koopman, Peter; Lovell-Badge, Robin

2017-01-01

During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the ~50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly.

Ionizing radiation enhances immunogenicity of cells expressing a tumor-specific T-cell epitope

International Nuclear Information System (INIS)

Ciernik, Ilja F.; Romero, Pedro; Berzofsky, Jay A.; Carbone, David P.

1999-01-01

Background: p53 point mutations represent potential tumor-specific cytolytic T lymphocyte (CTL) epitopes. Whether ionizing radiation (IR) alters the immunological properties of cells expressing mutant p53 in respect of the CTL epitope generated by a defined point mutation has not been evaluated. Methods: Mutant p53-expressing syngeneic, nontumor forming BALB/c 3T3 fibroblasts, tumor forming ras-transfected BALB/c 3T3 sarcomas, and DBA/2-derived P815 mastocytoma cells, which differ at the level of minor histocompatibility antigens, were used as cellular vaccines. Cells were either injected with or without prior IR into naive BALB/c mice. Cellular cytotoxicity was assessed after secondary restimulation of effector spleen cells in vitro. Results: Injection of P815 mastocytoma cells expressing the mutant p53 induced mutation-specific CTL in BALB/c mice irrespective of prior irradiation. However, syngeneic fibroblasts or fibrosarcomas endogenously expressing mutant p53 were able to induce significant mutation-specific CTL only when irradiated prior to injection into BALB/c mice. IR of fibroblasts did not detectably alter the expression of cell surface molecules involved in immune response induction, nor did it alter the short-term in vitro viability of the fibroblasts. Interestingly, radioactively-labeled fibroblasts injected into mice after irradiation showed altered organ distribution, suggesting that the in vivo fate of these cells may play a crucial role in their immunogenicity. Conclusions: These findings indicate that IR can alter the immunogenicity of syngeneic normal as well as tumor forming fibroblasts in vivo, and support the view that ionizing radiation enhances immunogenicity of cellular tumor vaccines

Synthetic cold-inducible promoter enhances recombinant protein accumulation during Agrobacterium-mediated transient expression in Nicotiana excelsior at chilling temperatures.

Science.gov (United States)

Gerasymenko, I M; Sheludko, Y V

2017-07-01

To exploit cold-inducible biochemical processes beneficial for foreign mRNA transcription, translation and storage, as well as protein product stability, during Agrobacterium-mediated transient expression. The efficiency of three different 5'-regulatory sequences to achieve transient expression of the GFP-based reporter gene under chilling conditions (6-8 °C since the 3rd day post inoculation) was compared. We studied the upstream sequences of a cold-inducible Arabidopsis thaliana cor15a gene, the core element of 35S CaMV promoter fused to the TMV omega 5'-UTR, and the synthetic promoter including the 35S core sequence and two binding sites for cold-inducible CBF transcription factors (P_DRE::35S). Cultivation of plants transiently expressing reporter gene under control of the synthetic P_DRE::35S promoter under chilling conditions since the 3rd dpi led to the reliably higher reporter accumulation as compared to the other tested regulatory sequences under chilling or greenhouse conditions. Reporter protein fluorescence under chilling conditions using P_DRE::35S reached 160% as compared to the transient expression in the greenhouse. Period of transient expression considerably extended if plants were cultivated at chilling temperature since the 3rd dpi: reporter protein fluorescence reached its maximum at the 20th dpi and was detected in leaves up to the 65th dpi. The enhanced protein accumulation at low temperature was accompanied by the prolonged period of corresponding mRNA accumulation. Transient expression under chilling conditions using synthetic cold-inducible promoter enhances target protein accumulation and may decrease greenhouse heating expenses.

Captodiamine, a putative antidepressant, enhances hypothalamic BDNF expression in vivo by synergistic 5-HT2c receptor antagonism and sigma-1 receptor agonism.

Science.gov (United States)

Ring, Rebecca M; Regan, Ciaran M

2013-10-01

The putative antidepressant captodiamine is a 5-HT2c receptor antagonist and agonist at sigma-1 and D3 dopamine receptors, exerts an anti-immobility action in the forced swim paradigm, and enhances dopamine turnover in the frontal cortex. Captodiamine has also been found to ameliorate stress-induced anhedonia, reduce the associated elevations of hypothalamic corticotrophin-releasing factor (CRF) and restore the reductions in hypothalamic BDNF expression. Here we demonstrate chronic administration of captodiamine to have no significant effect on hypothalamic CRF expression through sigma-1 receptor agonism; however, both sigma-1 receptor agonism or 5-HT2c receptor antagonism were necessary to enhance BDNF expression. Regulation of BDNF expression by captodiamine was associated with increased phosphorylation of transcription factor CREB and mediated through sigma-1 receptor agonism but blocked by 5-HT2c receptor antagonism. The existence of two separate signalling pathways was confirmed by immunolocalisation of each receptor to distinct cell populations in the paraventricular nucleus of the hypothalamus. Increased BDNF induced by captodiamine was also associated with enhanced expression of synapsin, but not PSD-95, suggesting induction of long-term structural plasticity between hypothalamic synapses. These unique features of captodiamine may contribute to its ability to ameliorate stress-induced anhedonia as the hypothalamus plays a prominent role in regulating HPA axis activity.

A single enhancer regulating the differential expression of duplicated red-sensitive opsin genes in zebrafish.

Directory of Open Access Journals (Sweden)

Taro Tsujimura

2010-12-01

Full Text Available A fundamental step in the evolution of the visual system is the gene duplication of visual opsins and differentiation between the duplicates in absorption spectra and expression pattern in the retina. However, our understanding of the mechanism of expression differentiation is far behind that of spectral tuning of opsins. Zebrafish (Danio rerio have two red-sensitive cone opsin genes, LWS-1 and LWS-2. These genes are arrayed in a tail-to-head manner, in this order, and are both expressed in the long member of double cones (LDCs in the retina. Expression of the longer-wave sensitive LWS-1 occurs later in development and is thus confined to the peripheral, especially ventral-nasal region of the adult retina, whereas expression of LWS-2 occurs earlier and is confined to the central region of the adult retina, shifted slightly to the dorsal-temporal region. In this study, we employed a transgenic reporter assay using fluorescent proteins and P1-artificial chromosome (PAC clones encompassing the two genes and identified a 0.6-kb "LWS-activating region" (LAR upstream of LWS-1, which regulates expression of both genes. Under the 2.6-kb flanking upstream region containing the LAR, the expression pattern of LWS-1 was recapitulated by the fluorescent reporter. On the other hand, when LAR was directly conjugated to the LWS-2 upstream region, the reporter was expressed in the LDCs but also across the entire outer nuclear layer. Deletion of LAR from the PAC clones drastically lowered the reporter expression of the two genes. These results suggest that LAR regulates both LWS-1 and LWS-2 by enhancing their expression and that interaction of LAR with the promoters is competitive between the two genes in a developmentally restricted manner. Sharing a regulatory region between duplicated genes could be a general way to facilitate the expression differentiation in duplicated visual opsins.

Expression and Purification of Glycosyltransferases in Pichia Pastoris: Towards Improving the Migration of Stem Cells by Enhancing Surface Expression of Sialyl Lewis X

KAUST Repository

Al-Amoodi, Asma S.

2017-05-01

Recruitment of circulating cells towards target sites is primarily dependent on E-selectin receptor/ligand adhesive interactions. Glycosyltransferase (GTs) are involved in the creation of E-selectin ligands. A sialofucosylated terminal tetrasaccharide like glycan structure known as sialyl Lewis x (sLex), is the most recognized ligand by selectins. This structure is found on the surface of cancer cells and leukocytes but is often absent on the surface of many adult stem cell populations. In order to synthesize sLex, GTs must be endogenously expressed and remain active within the cells. Generally, these stem cells express terminal sialylated lactosamine structures on their glycoproteins which require the addition of alpha-(1,3)-fucose to be converted into an E-selectin ligand. There are a number of fucosyltransferases (FUTs) that are able to modify terminal lactosamine structures to create sLex such as FUT6. In this work we focused on expressing and purifying active recombinant FUTs as a tool to help create sLex structures on the surface of adult stem cells in order to enhance their migration.

A novel mammal-specific three partite enhancer element regulates node and notochord-specific Noto expression.

Directory of Open Access Journals (Sweden)

Leonie Alten

Full Text Available The vertebrate organizer and notochord have conserved, essential functions for embryonic development and patterning. The restricted expression of developmental regulators in these tissues is directed by specific cis-regulatory modules (CRMs whose sequence conservation varies considerably. Some CRMs have been conserved throughout vertebrates and likely represent ancestral regulatory networks, while others have diverged beyond recognition but still function over a wide evolutionary range. Here we identify and characterize a mammalian-specific CRM required for node and notochord specific (NNC expression of NOTO, a transcription factor essential for node morphogenesis, nodal cilia movement and establishment of laterality in mouse. A 523 bp enhancer region (NOCE upstream the Noto promoter was necessary and sufficient for NNC expression from the endogenous Noto locus. Three subregions in NOCE together mediated full activity in vivo. Binding sites for known transcription factors in NOCE were functional in vitro but dispensable for NOCE activity in vivo. A FOXA2 site in combination with a novel motif was necessary for NOCE activity in vivo. Strikingly, syntenic regions in non-mammalian vertebrates showed no recognizable sequence similarities. In contrast to its activity in mouse NOCE did not drive NNC expression in transgenic fish. NOCE represents a novel, mammal-specific CRM required for the highly restricted Noto expression in the node and nascent notochord and thus regulates normal node development and function.

A novel mammal-specific three partite enhancer element regulates node and notochord-specific Noto expression.

Science.gov (United States)

Alten, Leonie; Schuster-Gossler, Karin; Eichenlaub, Michael P; Wittbrodt, Beate; Wittbrodt, Joachim; Gossler, Achim

2012-01-01

The vertebrate organizer and notochord have conserved, essential functions for embryonic development and patterning. The restricted expression of developmental regulators in these tissues is directed by specific cis-regulatory modules (CRMs) whose sequence conservation varies considerably. Some CRMs have been conserved throughout vertebrates and likely represent ancestral regulatory networks, while others have diverged beyond recognition but still function over a wide evolutionary range. Here we identify and characterize a mammalian-specific CRM required for node and notochord specific (NNC) expression of NOTO, a transcription factor essential for node morphogenesis, nodal cilia movement and establishment of laterality in mouse. A 523 bp enhancer region (NOCE) upstream the Noto promoter was necessary and sufficient for NNC expression from the endogenous Noto locus. Three subregions in NOCE together mediated full activity in vivo. Binding sites for known transcription factors in NOCE were functional in vitro but dispensable for NOCE activity in vivo. A FOXA2 site in combination with a novel motif was necessary for NOCE activity in vivo. Strikingly, syntenic regions in non-mammalian vertebrates showed no recognizable sequence similarities. In contrast to its activity in mouse NOCE did not drive NNC expression in transgenic fish. NOCE represents a novel, mammal-specific CRM required for the highly restricted Noto expression in the node and nascent notochord and thus regulates normal node development and function.

Arsenite enhances tumor necrosis factor-α-induced expression of vascular cell adhesion molecule-1

International Nuclear Information System (INIS)

Tsou, T.-C.; Yeh, Szu Ching; Tsai, E.-M.; Tsai, F.-Y.; Chao, H.-R.; Chang, Louis W.

2005-01-01

Epidemiological studies demonstrated a high association of vascular diseases with arsenite exposure. We hypothesize that arsenite potentiates the effect of proinflammatory cytokines on vascular endothelial cells, and hence contributes to atherosclerosis. In this study, we investigated the effect of arsenite and its induction of glutathione (GSH) on vascular cell adhesion molecule-1 (VCAM-1) protein expression in human umbilical vein endothelial cells (HUVECs) in response to tumor necrosis factor-α (TNF-α), a typical proinflammatory cytokine. Our study demonstrated that arsenite pretreatment potentiated the TNF-α-induced VCAM-1 expression with up-regulations of both activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). To elucidate the role of GSH in regulation of AP-1, NF-κB, and VCAM-1 expression, we employed L-buthionine (S,R)-sulfoximine (BSO), a specific γ-glutamylcysteine synthetase (γ-GCS) inhibitor, to block intracellular GSH synthesis. Our investigation revealed that, by depleting GSH, arsenite attenuated the TNF-α-induced VCAM-1 expression as well as a potentiation of AP-1 and an attenuation of NF-κB activations by TNF-α. Moreover, we found that depletion of GSH would also attenuate the TNF-α-induced VCAM-1 expression with a down-regulation of the TNF-α-induced NF-κB activation and without significant effect on AP-1. On the other hand, the TNF-α-induced VCAM-1 expression could be completely abolished by inhibition of AP-1 or NF-κB activity, suggesting that activation of both AP-1 and NF-κB was necessary for VCAM-1 expression. In summary, we demonstrate that arsenite enhances the TNF-α-induced VCAM-1 expression in HUVECs via regulation of AP-1 and NF-κB activities in a GSH-sensitive manner. Our present study suggested a potential mechanism for arsenite in the induction of vascular inflammation and vascular diseases via modulating the actions of proinflammatory cytokines

Enhanced tolerance and remediation of anthracene by transgenic tobacco plants expressing a fungal glutathione transferase gene

Energy Technology Data Exchange (ETDEWEB)

Dixit, Prachy; Mukherjee, Prasun K.; Sherkhane, Pramod D.; Kale, Sharad P. [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Eapen, Susan, E-mail: eapenhome@yahoo.com [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085 (India)

2011-08-15

Highlights: {yields} Transgenic plants expressing a TvGST gene were tested for tolerance, uptake and degradation of anthracene. {yields} Transgenic plants were more tolerant to anthracene and take up more anthracene from soil and solutions compared to control plants. {yields} Using in vitro T{sub 1} seedlings, we showed that anthracene-a three fused benzene ring compound was phytodegraded to naphthalene derivatives, having two benzene rings. {yields} This is the first time that a transgenic plant was shown to have the potential to phytodegrade anthracene. - Abstract: Plants can be used for remediation of polyaromatic hydrocarbons, which are known to be a major concern for human health. Metabolism of xenobiotic compounds in plants occurs in three phases and glutathione transferases (GST) mediate phase II of xenobiotic transformation. Plants, although have GSTs, they are not very efficient for degradation of exogenous recalcitrant xenobiotics including polyaromatic hydrocarbons. Hence, heterologous expression of efficient GSTs in plants may improve their remediation and degradation potential of xenobiotics. In the present study, we investigated the potential of transgenic tobacco plants expressing a Trichoderma virens GST for tolerance, remediation and degradation of anthracene-a recalcitrant polyaromatic hydrocarbon. Transgenic plants with fungal GST showed enhanced tolerance to anthracene compared to control plants. Remediation of {sup 14}C uniformly labeled anthracene from solutions and soil by transgenic tobacco plants was higher compared to wild-type plants. Transgenic plants (T{sub 0} and T{sub 1}) degraded anthracene to naphthalene derivatives, while no such degradation was observed in wild-type plants. The present work has shown that in planta expression of a fungal GST in tobacco imparted enhanced tolerance as well as higher remediation potential of anthracene compared to wild-type plants.

Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone.

Directory of Open Access Journals (Sweden)

Nitzan Gonen

2017-01-01

Full Text Available During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES, which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the ~50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly.

Dual expression of hTERT and VEGF prolongs life span and enhances angiogenic ability of aged BMSCs

Energy Technology Data Exchange (ETDEWEB)

Tang, Hao [Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou (China); Department of Neurosurgery, Affiliated Bayi Brain Hospital, The Military General Hospital of Beijing PLA, Beijing (China); Xiang, Yongsheng [Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou (China); Department of Neurosurgery, First Affiliated Hospital of Jinan University, Guangzhou (China); Jiang, Xiaodan; Ke, Yiquan; Xiao, Zongyu; Guo, Yang; Wang, Qiujing; Du, Mouxuan; Qin, Linsha; Zou, Yuxi; Cai, Yingqian [Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou (China); Chen, Zhenzhou, E-mail: czz1020@163.com [Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou (China); Xu, Ruxiang, E-mail: zjxuruxiang@163.com [Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou (China); Department of Neurosurgery, Affiliated Bayi Brain Hospital, The Military General Hospital of Beijing PLA, Beijing (China)

2013-11-01

Highlights: •Expression of hTERT and VEGF changed the lifespan and morphology of hBMSCs. •The expression of VEGF and hTRET promoted angiogenesis in vitro and in vivo. •The expression of VEGF and hTRET in hBMSCs had few effects on tumorigenicity. -- Abstract: Previous studies have confirmed the therapeutic effects of bone marrow stromal cells (BMSCs) transplantation on cerebral ischemia. However, the proliferative, differentiative, and homing capacity of BMSC from the elderly are significantly reduced, especially after several passages expansion in vitro. In this study, by introducing lentivirus-mediated hTERT and VEGF genes to modify human BMSCs from aged donors, we observed extended lifespan, promoted angiogenic capacity while less enhanced tumorigenicity of the genetically engineering BMSCs. These results therefore suggest that the modification of aged BMSCs by dual expression of hTERT and VEGF may be used for autologous cell replacement for ischemic cerebrovascular disease in elderly patients.

Dual expression of hTERT and VEGF prolongs life span and enhances angiogenic ability of aged BMSCs

International Nuclear Information System (INIS)

Tang, Hao; Xiang, Yongsheng; Jiang, Xiaodan; Ke, Yiquan; Xiao, Zongyu; Guo, Yang; Wang, Qiujing; Du, Mouxuan; Qin, Linsha; Zou, Yuxi; Cai, Yingqian; Chen, Zhenzhou; Xu, Ruxiang

2013-01-01

Highlights: •Expression of hTERT and VEGF changed the lifespan and morphology of hBMSCs. •The expression of VEGF and hTRET promoted angiogenesis in vitro and in vivo. •The expression of VEGF and hTRET in hBMSCs had few effects on tumorigenicity. -- Abstract: Previous studies have confirmed the therapeutic effects of bone marrow stromal cells (BMSCs) transplantation on cerebral ischemia. However, the proliferative, differentiative, and homing capacity of BMSC from the elderly are significantly reduced, especially after several passages expansion in vitro. In this study, by introducing lentivirus-mediated hTERT and VEGF genes to modify human BMSCs from aged donors, we observed extended lifespan, promoted angiogenic capacity while less enhanced tumorigenicity of the genetically engineering BMSCs. These results therefore suggest that the modification of aged BMSCs by dual expression of hTERT and VEGF may be used for autologous cell replacement for ischemic cerebrovascular disease in elderly patients

Lobular carcinoma in situ and invasive lobular breast cancer are characterized by enhanced expression of transcription factor AP-2β.

Science.gov (United States)

Raap, Mieke; Gronewold, Malte; Christgen, Henriette; Glage, Silke; Bentires-Alj, Mohammad; Koren, Shany; Derksen, Patrick W; Boelens, Mirjam; Jonkers, Jos; Lehmann, Ulrich; Feuerhake, Friedrich; Kuehnle, Elna; Gluz, Oleg; Kates, Ronald; Nitz, Ulrike; Harbeck, Nadia; Kreipe, Hans H; Christgen, Matthias

2018-01-01

Transcription factor AP-2β (TFAP2B) regulates embryonic organ development and is overexpressed in alveolar rhabdomyosarcoma, a rare childhood malignancy. Gene expression profiling has implicated AP-2β in breast cancer (BC). This study characterizes AP-2β expression in the mammary gland and in BC. AP-2β protein expression was assessed in the normal mammary gland epithelium, in various reactive, metaplastic and pre-invasive neoplastic lesions and in two clinical BC cohorts comprising >2000 patients. BCs from various genetically engineered mouse (GEM) models were also evaluated. Human BC cell lines served as functional models to study siRNA-mediated inhibition of AP-2β. The normal mammary gland epithelium showed scattered AP-2β-positive cells in the luminal cell layer. Various reactive and pre-invasive neoplastic lesions, including apocrine metaplasia, usual ductal hyperplasia and lobular carcinoma in situ (LCIS) showed enhanced AP-2β expression. Cases of ductal carcinoma in situ (DCIS) were more often AP-2β-negative (Pinvasive BC cohorts, AP-2β-positivity was associated with the lobular BC subtype (Plobular BC cell lines in vitro. In summary, AP-2β is a new mammary epithelial differentiation marker. Its expression is preferentially retained and enhanced in LCIS and invasive lobular BC and has prognostic implications. Our findings indicate that AP-2β controls tumor cell proliferation in this slow-growing BC subtype.

Bivalent promoter marks and a latent enhancer may prime the leukaemia oncogene LMO1 for ectopic expression in T-cell leukaemia.

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Oram, S H; Thoms, J; Sive, J I; Calero-Nieto, F J; Kinston, S J; Schütte, J; Knezevic, K; Lock, R B; Pimanda, J E; Göttgens, B

2013-06-01

LMO1 is a transcriptional regulator and a T-acute lymphoblastic leukaemia (T-ALL) oncogene. Although first identified in association with a chromosomal translocation in T-ALL, the ectopic expression of LMO1 occurs far more frequently in the absence of any known mutation involving its locus. Given that LMO1 is barely expressed in any haematopoietic lineage, and activation of transcriptional drivers in leukaemic cells is not well described, we investigated the regulation of this gene in normal haematopoietic and leukaemic cells. We show that LMO1 has two promoters that drive reporter gene expression in transgenic mice to neural tissues known to express endogenous LMO1. The LMO1 promoters display bivalent histone marks in multiple blood lineages including T-cells, and a 3' flanking region at LMO1 +57 contains a transcriptional enhancer that is active in developing blood cells in transgenic mouse embryos. The LMO1 promoters become activated in T-ALL together with the 3' enhancer, which is bound in primary T-ALL cells by SCL/TAL1 and GATA3. Taken together, our results show that LMO1 is poised for expression in normal progenitors, where activation of SCL/TAL1 together with a breakdown of epigenetic repression of LMO1 regulatory elements induces ectopic LMO1 expression that contributes to the development and maintenance of T-ALL.

Two estrogen response element sequences near the PCNA gene are not responsible for its estrogen-enhanced expression in MCF7 cells.

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Cheng Wang

Full Text Available The proliferating cell nuclear antigen (PCNA is an essential component of DNA replication, cell cycle regulation, and epigenetic inheritance. High expression of PCNA is associated with poor prognosis in patients with breast cancer. The 5'-region of the PCNA gene contains two computationally-detected estrogen response element (ERE sequences, one of which is evolutionarily conserved. Both of these sequences are of undocumented cis-regulatory function. We recently demonstrated that estradiol (E2 enhances PCNA mRNA expression in MCF7 breast cancer cells. MCF7 cells proliferate in response to E2.Here, we demonstrate that E2 rapidly enhanced PCNA mRNA and protein expression in a process that requires ERalpha as well as de novo protein synthesis. One of the two upstream ERE sequences was specifically bound by ERalpha-containing protein complexes, in vitro, in gel shift analysis. Yet, each ERE sequence, when cloned as a single copy, or when engineered as two tandem copies of the ERE-containing sequence, was not capable of activating a luciferase reporter construct in response to E2. In MCF7 cells, neither ERE-containing genomic region demonstrated E2-dependent recruitment of ERalpha by sensitive ChIP-PCR assays.We conclude that E2 enhances PCNA gene expression by an indirect process and that computational detection of EREs, even when evolutionarily conserved and when near E2-responsive genes, requires biochemical validation.

Nimotuzumab enhances temozolomide-induced growth suppression of glioma cells expressing mutant EGFR in vivo

International Nuclear Information System (INIS)

Nitta, Yusuke; Shimizu, Saki; Shishido-Hara, Yukiko; Suzuki, Kaori; Shiokawa, Yoshiaki; Nagane, Motoo

2016-01-01

A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti-EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild-type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and phosphorylation status of molecules were determined by Western blot analysis. Methylation status of promoter region of O 6 -methylguanine-DNA methyltransferase (MGMT) was detected by methylation-specific PCR. Antitumor activity was tested using nude mice bearing either subcutaneous or intracerebral xenografts along with analyses of EGFR phosphorylation status, proliferation, apoptosis, and vessel density. Nimotuzumab treatment resulted in reduction of EGFRvIII tyrosine phosphorylation with a decrease in Akt phosphorylation that was greater than that of wtEGFR. Correspondingly, antitumor effects, growth suppression and survival elongation, were more significant in mice bearing either subcutaneous or intracerebral tumor expressing EGFRvIII than in those expressing wtEGFR. These effects were markedly increased when temozolomide was combined with nimotuzumab. The post-treatment recurrent brain tumors exhibited a decrease in expression of the mismatch repair (MMR) proteins, MSH6 and MLH1, but their methylated MGMT status did not changed. Nimotuzumab has in vivo antitumor activity against GBM, especially those expressing EGFRvIII, when combined with temozolomide. This could provide a basis for preselection of patients with GBM by EGFR status who might benefit from the nimotuzumab and temozolomide combination therapy

Identification of Variants in RET and IHH Pathway Members in a Large Family With History of Hirschsprung Disease.

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Sribudiani, Y; Chauhan, R K; Alves, M M; Petrova, L; Brosens, E; Harrison, C; Wabbersen, T; de Graaf, B M; Rügenbrink, T; Burzynski, G; Brouwer, R W W; IJcken, W F J van; Maas, S M; de Klein, A; Osinga, J; Eggen, B J L; Burns, A J; Brooks, A S; Shepherd, I T; Hofstra, R M W

2018-03-27

Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multi-generational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3. However, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. Within the linkage region, we identified 1 putative splice variant in the LPS responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on mRNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the indian hedgehog gene (IHH) and its mediator

Curcumin enhances the mitomycin C-induced cytotoxicity via downregulation of MKK1/2-ERK1/2-mediated Rad51 expression in non-small cell lung cancer cells

International Nuclear Information System (INIS)

Ko, Jen-Chung; Tsai, Min-Shao; Weng, Shao-Hsing; Kuo, Ya-Hsun; Chiu, Yu-Fan; Lin, Yun-Wei

2011-01-01

Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC) cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2-ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC. - Highlights: → Curcumin downregulates MKK-ERK-mediated Rad51 expression. → Curcumin enhances mitomycin C-induced cytotoxicity. → Rad51 protects cells from cytotoxic effects induced by curcumin and mitomycin C. → Rad51 inhibition enhances the chemosensitization of

High hydrostatic pressure activates gene expression that leads to ethanol production enhancement in a Saccharomyces cerevisiae distillery strain

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Bravim, Fernanda; Lippman, Soyeon I.; da Silva, Lucas F.; Souza, Diego T.; Fernandes, A. Alberto R.; Masuda, Claudio A.; Broach, James R.

2016-01-01

High hydrostatic pressure (HHP) is a stress that exerts broad effects on microorganisms with characteristics similar to those of common environmental stresses. In this study, we aimed to identify genetic mechanisms that can enhance alcoholic fermentation of wild Saccharomyces cerevisiae isolated from Brazilian spirit fermentation vats. Accordingly, we performed a time course microarray analysis on a S. cerevisiae strain submitted to mild sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min without pressure treatment. The obtained transcriptional profiles demonstrate the importance of post-pressurisation period on the activation of several genes related to cell recovery and stress tolerance. Based on these results, we over-expressed genes strongly induced by HHP in the same wild yeast strain and identified genes, particularly SYM1, whose over-expression results in enhanced ethanol production and stress tolerance upon fermentation. The present study validates the use of HHP as a biotechnological tool for the fermentative industries. PMID:22915193

Biodesulfurization of dibenzothiophene in Escherichia coli is enhanced by expression of a Vibrio harveyi oxidoreductase gene

Energy Technology Data Exchange (ETDEWEB)

Reichmuth, D.S.; Hittle, J.L.; Blanch, H.W.; Keasling, J.D.

2000-01-05

One possible alternative to current fuel hydrodesulfurization methods is the use of microorganisms to remove sulfur compounds. Biodesulfurization requires much milder processing conditions, gives higher specificity, and does not require molecular hydrogen. In the present work the authors have produced two compatible plasmids: pDSR3, which allows Escherichia coli to convert dibenzothiophene (DBT) to hydroxybiphenyl (HBP), and pDSR2, which produces a Vibrio harveyi flavin oxidoreductase. The authors show that the flavin oxidoreductase enhances the rate of DBT removal when co-expressed in vivo with the desulfurization enzymes. The plasmids pDSR2 and pDSR3 were co-expressed in growing cultures. The expression of oxidoreductase caused an increase in the rate of DBT removal but a decrease in the rate of HBP production. The maximum rate of DBT removal was 8 mg/h {center{underscore}dot} g dry cell weight. Experiments were also conducted using resting cells with the addition of various carbon sources. It was found that the addition of glucose or glycerol to cultures with oxidoreductase expression produced the highest DBT removal rate. The culture with acetate and no oxidoreductase expression had the highest level of HBP production. For all carbon sources, the DBT removal rate was faster and the HBP generation rate slower with the expression of the oxidoreductase. Analysis of desulfurization intermediates indicates that the last enzyme in the pathway may be limiting.

The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1.

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Zhou, Xiangjun; Sun, Tian-Hu; Wang, Ning; Ling, Hong-Qing; Lu, Shan; Li, Li

2011-04-01

The cauliflower (Brassica oleracea var. botrytis) Orange (Or) gene affects plant growth and development in addition to conferring β-carotene accumulation. This study was undertaken to investigate the molecular basis for the effects of the Or gene mutation in on plant growth. The OR protein was found to interact with cauliflower and Arabidopsis eukaryotic release factor 1-2 (eRF1-2), a member of the eRF1 family, by yeast two-hybrid analysis and by bimolecular fluorescence complementation (BiFC) assay. Concomitantly, the Or mutant showed reduced expression of the BoeRF1 family genes. Transgenic cauliflower plants with suppressed expression of BoeRF1-2 and BoeRF1-3 were generated by RNA interference. Like the Or mutant, the BoeRF1 RNAi lines showed increased elongation of the leaf petiole. This long-petiole phenotype was largely caused by enhanced cell elongation, which resulted from increased cell length and elevated expression of genes involved in cell-wall loosening. These findings demonstrate that the cauliflower Or gene controls petiole elongation by suppressing the expression of eRF1 genes, and provide new insights into the molecular mechanism of leaf petiole regulation. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

c-Myc Enhances Sonic Hedgehog-Induced Medulloblastoma Formation from Nestin-Expressing Neural Progenitors in Mice

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Ganesh Rao

2003-05-01

Full Text Available Medulloblastomas are malignant brain tumors that arise in the cerebella of children. The presumed cellsof-origin are undifferentiated precursors of granule neurons that occupy the external granule layer (EGL of the developing cerebellum. The overexpression of proteins that normally stimulate proliferation of neural progenitor cells may initiate medulloblastoma formation. Two known mitogens for neural progenitors are the c-Myc oncoprotein and Sonic hedgehog (Shh, a crucial determinant of embryonic pattern formation in the central nervous system. We modeled the ability of c-Myc and Shh to induce medulloblastoma in mice using the RCAS/tv-a system, which allows postnatal gene transfer and expression in a cell type-specific manner. We targeted the expression of Shh and c-Myc to nestin-expressing neural progenitor cells by injecting replication-competent ALV splice acceptor (RCAS vectors into the cerebella of newborn mice. Following injection with RCAS-Shh alone, 3/32 (9% mice developed medulloblastomas and 5/32 showed multifocal hyperproliferation of the EGL, possibly a precursor stage of medulloblastoma. Following injection with RCAS-Shh plus RCAS-Myc, 9/39 (23% mice developed medulloblastomas. We conclude that nestin-expressing neural progenitors, present in the cerebellum at birth, can act as the cells-of-origin for medulloblastoma, and that c-Myc cooperates with Shh to enhance tumorigenicity.

Cerebrospinal fluid levels of glial cell-derived neurotrophic factor correlate with spinal cord stimulation frequency in patients with neuropathic pain: a preliminary report.

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McCarthy, K F; McCrory, C

2014-08-01

Case series. To evaluate relationships between spinal cord stimulation (SCS) parameters and levels of glial cell-derived neurotrophic factor (GDNF). Ambulatory pain clinic of St James's Hospital, Dublin, Ireland. Nine patients with an implanted SCS and Failed Back Surgery Syndrome (FBSS) were administered the Brief Pain Inventory and Short Form (36) Health Survey. Following a lumbar puncture, levels of GDNF in cerebrospinal fluid (CSF) were assayed and correlated with stimulation parameters. Controls were patients with arthritic back pain who were matched for age, gender and SF-36 score. Concentrations of GDNF in CSF are higher in patients with FBSS than controls (P=0.002) and correlate with SCS frequency (P=0.029). Concentrations of GDNF in CSF are higher in neuropathic pain and appear to be related to stimulation frequency. Further work is needed to evaluate this potential relationship, both in neuropathic pain and in other contexts such as locomotor dysfunction.

Zinc deficiency promotes cystitis-related bladder pain by enhancing function and expression of Cav3.2 in mice.

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Ozaki, Tomoka; Matsuoka, Junki; Tsubota, Maho; Tomita, Shiori; Sekiguchi, Fumiko; Minami, Takeshi; Kawabata, Atsufumi

2018-01-15

Ca v 3.2 T-type Ca 2+ channel activity is suppressed by zinc that binds to the extracellular histidine-191 of Ca v 3.2, and enhanced by H 2 S that interacts with zinc. Ca v 3.2 in nociceptors is upregulated in an activity-dependent manner. The enhanced Ca v 3.2 activity by H 2 S formed by the upregulated cystathionine-γ-lyase (CSE) is involved in the cyclophosphamide (CPA)-induced cystitis-related bladder pain in mice. We thus asked if zinc deficiency affects the cystitis-related bladder pain in mice by altering Ca v 3.2 function and/or expression. Dietary zinc deficiency for 2 weeks greatly decreased zinc concentrations in the plasma but not bladder tissue, and enhanced the bladder pain/referred hyperalgesia (BP/RH) following CPA at 200mg/kg, a subeffective dose, but not 400mg/kg, a maximal dose, an effect abolished by pharmacological blockade or gene silencing of Ca v 3.2. Acute zinc deficiency caused by systemic N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN), a zinc chelator, mimicked the dietary zinc deficiency-induced Ca v 3.2-dependent promotion of BP/RH following CPA at 200mg/kg. CPA at 400mg/kg alone or TPEN plus CPA at 200mg/kg caused Ca v 3.2 overexpression accompanied by upregulation of Egr-1 and USP5, known to promote transcriptional expression and reduce proteasomal degradation of Ca v 3.2, respectively, in the dorsal root ganglia (DRG). The CSE inhibitor, β-cyano-l-alanine, prevented the BP/RH and upregulation of Ca v 3.2, Egr-1 and USP5 in DRG following TPEN plus CPA at 200mg/kg. Together, zinc deficiency promotes bladder pain accompanying CPA-induced cystitis by enhancing function and expression of Ca v 3.2 in nociceptors, suggesting a novel therapeutic avenue for treatment of bladder pain, such as zinc supplementation. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancement of myocardial regeneration through genetic engineering of cardiac progenitor cells expressing Pim-1 kinase.

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Fischer, Kimberlee M; Cottage, Christopher T; Wu, Weitao; Din, Shabana; Gude, Natalie A; Avitabile, Daniele; Quijada, Pearl; Collins, Brett L; Fransioli, Jenna; Sussman, Mark A

2009-11-24

Despite numerous studies demonstrating the efficacy of cellular adoptive transfer for therapeutic myocardial regeneration, problems remain for donated cells with regard to survival, persistence, engraftment, and long-term benefits. This study redresses these concerns by enhancing the regenerative potential of adoptively transferred cardiac progenitor cells (CPCs) via genetic engineering to overexpress Pim-1, a cardioprotective kinase that enhances cell survival and proliferation. Intramyocardial injections of CPCs overexpressing Pim-1 were given to infarcted female mice. Animals were monitored over 4, 12, and 32 weeks to assess cardiac function and engraftment of Pim-1 CPCs with echocardiography, in vivo hemodynamics, and confocal imagery. CPCs overexpressing Pim-1 showed increased proliferation and expression of markers consistent with cardiogenic lineage commitment after dexamethasone exposure in vitro. Animals that received CPCs overexpressing Pim-1 also produced greater levels of cellular engraftment, persistence, and functional improvement relative to control CPCs up to 32 weeks after delivery. Salutary effects include reduction of infarct size, greater number of c-kit(+) cells, and increased vasculature in the damaged region. Myocardial repair is significantly enhanced by genetic engineering of CPCs with Pim-1 kinase. Ex vivo gene delivery to enhance cellular survival, proliferation, and regeneration may overcome current limitations of stem cell-based therapeutic approaches.

A New Synthetic Compound, 2-OH, Enhances Interleukin-2 and Interferon-γ Gene Expression in Human Peripheral Blood Mononuclear Cells

Directory of Open Access Journals (Sweden)

Woan-Fang Tzeng

2009-07-01

Full Text Available A new synthetic compound, 6-hydroxy-2-tosylisoquinolin-1(2H-one (2-OH, was selected for immunopharmacological activity tests. The effects of 2-OH on human peripheral blood mononuclear cell (PBMC proliferation were determined by tritiated thymidine uptake. Compared to phytohemagglutinin (PHA; 5 μg/mL stimulation, 2-OH significantly enhanced PBMC proliferation in a dose-dependent manner. The 50% enhancement activity (EC50 for 2-OH was 4.4±0.1 μM. In addition, effects of 2-OH on interleukin-2 (IL-2 and interferon-γ (IFN-γ production in PBMC were determined by enzyme immunoassay. Results demonstrated that 2-OH stimulated IL-2 and IFN-γ production in PBMC. Data from reverse transcription-polymerase chain reaction (RT-PCR and real-time PCR indicated that IL-2 and IFN-γ mRNA expression in PBMC could be induced by 2-OH. Therefore, 2-OH enhanced IL-2 and IFN-γ production in PBMC by modulation their gene expression. We suggest that 2-OH may be an immunomodulatory agent.

Expression of Na+/glucose co-transporter 1 (SGLT1) is enhanced by supplementation of the diet of weaning piglets with artificial sweeteners.

Science.gov (United States)

Moran, Andrew W; Al-Rammahi, Miran A; Arora, Daleep K; Batchelor, Daniel J; Coulter, Erin A; Daly, Kristian; Ionescu, Catherine; Bravo, David; Shirazi-Beechey, Soraya P

2010-09-01

In an intensive livestock production, a shorter suckling period allows more piglets to be born. However, this practice leads to a number of disorders including nutrient malabsorption, resulting in diarrhoea, malnutrition and dehydration. A number of strategies have been proposed to overcome weaning problems. Artificial sweeteners, routinely included in piglets' diet, were thought to enhance feed palatability. However, it is shown in rodent models that when included in the diet, they enhance the expression of Na+/glucose co-transporter (SGLT1) and the capacity of the gut to absorb glucose. Here, we show that supplementation of piglets' feed with a combination of artificial sweeteners saccharin and neohesperidin dihydrochalcone enhances the expression of SGLT1 and intestinal glucose transport function. Artificial sweeteners are known to act on the intestinal sweet taste receptor T1R2/T1R3 and its partner G-protein, gustducin, to activate pathways leading to SGLT1 up-regulation. Here, we demonstrate that T1R2, T1R3 and gustducin are expressed together in the enteroendocrine cells of piglet intestine. Furthermore, gut hormones secreted by the endocrine cells in response to dietary carbohydrates, glucagon-like peptides (GLP)-1, GLP-2 and glucose-dependent insulinotrophic peptide (GIP), are co-expressed with type 1 G-protein-coupled receptors (T1R) and gustducin, indicating that L- and K-enteroendocrine cells express these taste elements. In a fewer endocrine cells, T1R are also co-expressed with serotonin. Lactisole, an inhibitor of human T1R3, had no inhibitory effect on sweetener-induced SGLT1 up-regulation in piglet intestine. A better understanding of the mechanism(s) involved in sweetener up-regulation of SGLT1 will allow the identification of nutritional targets with implications for the prevention of weaning-related malabsorption.

Local delivery of glial cell line-derived neurotrophic factor improves facial nerve regeneration after late repair.

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Barras, Florian M; Kuntzer, Thierry; Zurn, Anne D; Pasche, Philippe

2009-05-01

Facial nerve regeneration is limited in some clinical situations: in long grafts, by aged patients, and when the delay between nerve lesion and repair is prolonged. This deficient regeneration is due to the limited number of regenerating nerve fibers, their immaturity and the unresponsiveness of Schwann cells after a long period of denervation. This study proposes to apply glial cell line-derived neurotrophic factor (GDNF) on facial nerve grafts via nerve guidance channels to improve the regeneration. Two situations were evaluated: immediate and delayed grafts (repair 7 months after the lesion). Each group contained three subgroups: a) graft without channel, b) graft with a channel without neurotrophic factor; and c) graft with a GDNF-releasing channel. A functional analysis was performed with clinical observation of facial nerve function, and nerve conduction study at 6 weeks. Histological analysis was performed with the count of number of myelinated fibers within the graft, and distally to the graft. Central evaluation was assessed with Fluoro-Ruby retrograde labeling and Nissl staining. This study showed that GDNF allowed an increase in the number and the maturation of nerve fibers, as well as the number of retrogradely labeled neurons in delayed anastomoses. On the contrary, after immediate repair, the regenerated nerves in the presence of GDNF showed inferior results compared to the other groups. GDNF is a potent neurotrophic factor to improve facial nerve regeneration in grafts performed several months after the nerve lesion. However, GDNF should not be used for immediate repair, as it possibly inhibits the nerve regeneration.

Gene expression of hematoregulatory cytokines is elevated endogenously after sublethal gamma irradiation and is differentially enhanced by therapeutic administration of biologic response modifiers

International Nuclear Information System (INIS)

Peterson, V.M.; Adamovicz, J.J.; Madonna, G.S.; Gause, W.C.; Elliott, T.B.; Moore, M.M.; Ledney, G.D.; Jackson, W.E. III

1994-01-01

Prompt, cytokine-mediated restoration of hematopoiesis is a prerequisite for survival after irradiation. Therapy with biologic response modifiers (BRMs), such as LPS, 3D monophosphoryl lipid A (MPL), and synthetic trehalose dicrynomycolate (S-TDCM) presumably accelerates hematopoietic recovery after irradiation are poorly defined. One hour after sublethal (7.0 Gy) 60 Co gamma irradiation, B6D2F1/J female mice received a single i.p. injection of LPS, MPL, S-TDCM, an extract from Serratia marcescens (Sm-BRM), or Tween 80 in saline (TS). Five hours later, a quantitative reverse transcription-PCR assay demonstrated marked splenic gene expression for IL-1β, IL-3, IL-6, and granulocyte-CSF (G-CSF). Enhanced gene expression for TNF-α, macrophage-CSF (M-CSF), and stem cell factor (SCF) was not detected. Injection of any BRM further enhanced cytokine gene expression and plasma levels of CSF activity within 24 h after irradiation and hastened bone marrow recovery. Mice injected with S-TDCM or Sm-BRM sustained expression of the IL-6 gene for at least 24 h after irradiation. Sm-BRM-treated mice exhibited greater gene expression for IL-1β, IL-3, TNF-α, and G-CSF at day 1 than any other BRM. When challenged with 2 LD 50/30 of Klebsiella pneumoniae 4 days after irradiation, 100% of Sm-BRM-treated mice and 70% of S-TDCM-treated mice survived, whereas ≤30% of mice treated with LPS, MPL, or TS survived. Thus, sublethal irradiation induces transient, splenic cytokine gene expression that can be differentially amplified and prolonged by BRMs. BRMs that sustained and/or enhanced irradiation-induced expression of specific cytokine genes improved survival after experimental infection. 67 refs., 7 figs., 1 tab

Enhancing expression of SSU1 genes in Saccharomyces uvarum leads to an increase in sulfite tolerance and a transcriptome profile change.

Science.gov (United States)

Liu, X Z; Sang, M; Zhang, X A; Zhang, T K; Zhang, H Y; He, X; Li, S X; Sun, X D; Zhang, Z M

2017-05-01

Saccharomyces uvarum is a good wine yeast species that may have great potential for the future. However, sulfur tolerance of most S. uvarum strains is very poor. In addition there is still little information about the SSU1 gene of S. uvarum, which encodes a putative transporter conferring sulfite tolerance. In order to analyze the function of the SSU1 gene, two expression vectors that contained different SSU1 genes were constructed and transferred into a sulfite-tolerant S. uvarum strain, A9. Then sulfite tolerance, SO2 production, and PCR, sequencing, RT-qPCR and transcriptome analyses were used to access the function of the S. uvarum SSU1 gene. Our results illustrated that enhancing expression of the SSU1 gene can promote sulfite resistance in S. uvarum, and an insertion fragment ahead of the additional SSU1 gene, as seen in some alleles, could affect the expression of other genes and the sulfite tolerance level of S. uvarum. This is the first report on enhancing the expression of the SSU1 gene of S. uvarum. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Enhanced fatty acid oxidation and FATP4 protein expression after endurance exercise training in human skeletal muscle

DEFF Research Database (Denmark)

Jeppesen, Jacob; Jordy, Andreas B; Sjøberg, Kim A

2012-01-01

; however, it is not known whether this involves up-regulation of FATP1 and FATP4 protein. Therefore, the aim of this project was to investigate FATP1 and FATP4 protein expression in the vastus lateralis muscle from healthy human individuals and to what extent FATP1 and FATP4 protein expression were......FATP1 and FATP4 appear to be important for the cellular uptake and handling of long chain fatty acids (LCFA). These findings were obtained from loss- or gain of function models. However, reports on FATP1 and FATP4 in human skeletal muscle are limited. Aerobic training enhances lipid oxidation...

Enhanced expressions of microvascular smooth muscle receptors after focal cerebral ischemia occur via the MAPK MEK/ERK pathway

DEFF Research Database (Denmark)

Maddahi, A.; Edvinsson, L.

2008-01-01

), the enhanced vascular receptor expression, and attenuated the cerebral infarct and improved neurology score. CONCLUSION: Our results show that MCAO results in upregulation of cerebrovascular ETB, AT1 and 5-HT1B receptors. Blockade of this event with a MEK1 inhibitor as late as 6 h after the insult reduced...... the role of the MEK/ERK pathway in receptor expression following ischemic brain injury using the specific MEK1 inhibitor U0126. METHODS AND RESULT: Rats were subjected to a 2-h middle cerebral artery occlusion (MCAO) followed by reperfusion for 48-h and the ischemic area was calculated. The expression...... of phosphorylated ERK1/2 and Elk-1, and of endothelin ETA and ETB, angiotensin AT1, and 5-hydroxytryptamine 5-HT1B receptors were analyzed with immunohistochemistry using confocal microscopy in cerebral arteries, microvessels and in brain tissue. The expression of endothelin ETB receptor was analyzed...

Fractal structure in the volumetric contrast enhancement of malignant gliomas as a marker of oxidative metabolic pathway gene expression

NARCIS (Netherlands)

Miller, Kai J.; Berendsen, Sharon; Seute, Tatjana; Yeom, Kristen; Gephardt, Melanie H.; Grant, Gerald A.; Robe, Pierre A.

2017-01-01

Background: Fractal structure is found throughout many processes in nature, and often arises from sets of simple rules. We examined MRI contrast enhancement patterns from glioblastoma patients for evidence of fractal structure and correlated these with gene expression patterns. Methods: For 39

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

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Hacker, David L; Bertschinger, Martin; Baldi, Lucia; Wurm, Florian M

2004-10-27

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

Enhanced expression in vivo of HLA-ABC antigens and beta 2-microglobulin on human lymphoid cells induced by human interferon-alpha in patients with lung cancer. Enhanced expression of class I major histocompatibility antigens prior to treatment

DEFF Research Database (Denmark)

Nissen, Mogens Holst; Plesner, T; Larsen, J K

1985-01-01

than 0.5, respectively) by day-to-day analysis of an untreated healthy control group. An increased expression of both HLA-ABC (mean 55%, P less than 0.0005) and beta 2m (mean 23%, P less than 0.01) was also observed prior to treatment in the lung cancer patients when compared to a group of age matched......The effect of cloned human interferon-alpha (IFN-alpha) on the expression of HLA-ABC antigens (HLA-ABC) and beta 2-microglobulin (beta 2m) on human peripheral lymphoid cells in vivo was studied by cytofluorometry using monoclonal antibodies and fluorescein-labelled rabbit anti-mouse immunoglobulin....... A significant increase in the mean fluorescence intensity of HLA-ABC (median 59%, P less than 0.001) and beta 2m (median 57%, P less than 0.001) on small lymphoid cells was observed 24 h after initiation of IFN-alpha treatment (50 X 10(6) units IFN-alpha/m2 three times a week). The enhanced expression...

Ribavirin enhances IFN-α signalling and MxA expression: a novel immune modulation mechanism during treatment of HCV.

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Nigel J Stevenson

Full Text Available The nucleoside analogue Ribavirin significantly increases patient response to IFN-α treatment of HCV, by directly inhibiting viral replication. Recent studies indicate that Ribavirin also regulates immunity and we propose that Ribavirin enhances specific interferon sensitive gene (ISG expression by amplifying the IFN-α-JAK/STAT pathway. We found that IFN-α-induced STAT1 and STAT3 phosphorylation was increased in hepatocytes co-treated with Ribavirin and IFN-α, compared to IFN-α alone. Ribavirin specifically enhanced IFN-α induced mRNA and protein of the anti-viral mediator MxA, which co-localised with HCV core protein. These novel findings indicate for the first time that Ribavirin, in addition to its viral incorporation, also enhances IFN-α-JAK/STAT signalling, leading to a novel MxA-mediated immuno-modulatory mechanism that may enhance IFN-α anti-viral activity against HCV.

Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins.

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Frederix, Marijke; Edwards, Anne; Swiderska, Anna; Stanger, Andrew; Karunakaran, Ramakrishnan; Williams, Alan; Abbruscato, Pamela; Sanchez-Contreras, Maria; Poole, Philip S; Downie, J Allan

2014-08-01

In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Enhanced cerebrovascular expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 via the MEK/ERK pathway during cerebral ischemia in the rat

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Maddahi, Aida; Chen, Qingwen; Edvinsson, Lars

2009-01-01

BACKGROUND: Cerebral ischemia is usually characterized by a reduction in local blood flow and metabolism and by disruption of the blood-brain barrier in the infarct region. The formation of oedema and opening of the blood-brain barrier in stroke is associated with enhanced expression of metallopr......BACKGROUND: Cerebral ischemia is usually characterized by a reduction in local blood flow and metabolism and by disruption of the blood-brain barrier in the infarct region. The formation of oedema and opening of the blood-brain barrier in stroke is associated with enhanced expression...

Gata4 expression in lateral mesoderm is downstream of BMP4 and isactivated directly by Forkhead and GATA transcription factors through adistal enhancer element

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Rojas, Anabel; De Val, Sarah; Heidt, Analeah B.; Xu, Shan-Mei; Bristow, James; Black, Brian L.

2005-05-20

The GATA family of zinc-finger transcription factors plays key roles in the specification and differentiation of multiple cell types during development. GATA4 is an early regulator of gene expression during the development of endoderm and mesoderm, and genetic studies in mice have demonstrated that GATA4 is required for embryonic development.Despite the importance of GATA4 in tissue specification and differentiation, the mechanisms by which Gata4 expression is activated and the transcription factor pathways upstream of GATA4 remain largely undefined. To identify transcriptional regulators of Gata4 in the mouse,we screened conserved noncoding sequences from the mouse Gata4 gene for enhancer activity in transgenic embryos. Here, we define the regulation of a distal enhancer element from Gata4 that is sufficient to direct expression throughout the lateral mesoderm, beginning at 7.5 days of mouse embryonic development. The activity of this enhancer is initially broad but eventually becomes restricted to the mesenchyme surrounding the liver. We demonstrate that the function of this enhancer in transgenic embryos is dependent upon highly conserved Forkhead and GATA transcription factor binding sites, which are bound by FOXF1 and GATA4,respectively. Furthermore, the activity of the Gata4 lateral mesoderm enhancer is attenuated by the BMP antagonist Noggin, and the enhancer is not activated in Bmp4-null embryos. Thus, these studies establish that Gata4 is a direct transcriptional target of Forkhead and GATA transcription factors in the lateral mesoderm, and demonstrate that Gata4lateral mesoderm enhancer activation requires BMP4, supporting a model in which GATA4 serves as a downstream effector of BMP signaling in the lateral mesoderm.

Enhanced cerebrovascular expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 via the MEK/ERK pathway during cerebral ischemia in the rat

DEFF Research Database (Denmark)

Maddahi, Aida; Chen, Qingwen; Edvinsson, Lars

2009-01-01

. Immunocytochemistry showed no overlap in expression between MMP-9/TIMP-1 and the astrocyte/glial cell marker GFAP in the vessel walls. CONCLUSION: These data are the first to show that the elevated vascular expression of MMP-9 and TIMP-1, associated with breakdown of the blood-brain barrier following focal ischemia......BACKGROUND: Cerebral ischemia is usually characterized by a reduction in local blood flow and metabolism and by disruption of the blood-brain barrier in the infarct region. The formation of oedema and opening of the blood-brain barrier in stroke is associated with enhanced expression...... microscopy revealed enhanced expression of MMP-9, TIMP-1, and phosphorylated ERK1/2 in the smooth muscle cells of the ischemic MCA and associated intracerebral microvessels. The specific MEK1/2 inhibitor U0126, given intraperitoneal zero or 6 hours after the ischemic event, reduced the infarct volume...

Biodesulfurization of dibenzothiophene in Escherichia coli is enhanced by expression of a Vibrio harveyi oxidoreductase gene.

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Reichmuth, D S; Hittle, J L; Blanch, H W; Keasling, J D

2000-01-05

One possible alternative to current fuel hydrodesulfurization methods is the use of microorganisms to remove sulfur compounds. Biodesulfurization requires much milder processing conditions, gives higher specificity, and does not require molecular hydrogen. In the present work we have produced two compatible plasmids: pDSR3, which allows Escherichia coli to convert dibenzothiophene (DBT) to hydroxybiphenyl (HBP), and pDSR2, which produces a Vibrio harveyi flavin oxidoreductase. We show that the flavin oxidoreductase enhances the rate of DBT removal when co-expressed in vivo with the desulfurization enzymes. The plasmids pDSR2 and pDSR3 were co-expressed in growing cultures. The expression of oxidoreductase caused an increase in the rate of DBT removal but a decrease in the rate of HBP production. The maximum rate of DBT removal was 8 mg/h. g dry cell weight. Experiments were also conducted using resting cells with the addition of various carbon sources. It was found that the addition of glucose or glycerol to cultures with oxidoreductase expression produced the highest DBT removal rate (51 mg/h. g dry cell weight). The culture with acetate and no oxidoreductase expression had the highest level of HBP production. For all carbon sources, the DBT removal rate was faster and the HBP generation rate slower with the expression of the oxidoreductase. Analysis of desulfurization intermediates indicates that the last enzyme in the pathway may be limiting. Copyright 2000 John Wiley & Sons, Inc.

Long-time treatment by low-dose N-acetyl-L-cysteine enhances proinflammatory cytokine expressions in LPS-stimulated macrophages.

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Tomokazu Ohnishi

Full Text Available N-acetyl-L-cysteine is known to act as a reactive oxygen species scavenger and used in clinical applications. Previous reports have shown that high-dose N-acetyl-L-cysteine treatment inhibits the expression of proinflammatory cytokines in activated macrophages. Here, we have found that long-time N-acetyl-L-cysteine treatment at low-concentration increases phosphorylation of extracellular signal-regulated kinase 1/2 and AKT, which are essential for the induction of proinflammatory cytokines including interleukin 1β and interleukin 6 in lipopolysaccharide-stimulated RAW264.7 cells. Furthermore, long-time N-acetyl-L-cysteine treatment decreases expressions of protein phosphatases, catalytic subunit of protein phosphatase-2A and dual specificity phosphatase 1. On the other hand, we have found that short-time N-acetyl-L-cysteine treatment at low dose increases p53 expression, which inhibits expressions of proinflammatory cytokines. These observations suggest that long-time low-dose N-acetyl-L-cysteine treatment increases expressions of proinflammatory cytokines through enhancement of kinase phosphorylation.

Cytotoxic effects of replication-competent adenoviruses on human esophageal carcinoma are enhanced by forced p53 expression

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Yang, Shan; Kawamura, Kiyoko; Okamoto, Shinya; Yamauchi, Suguru; Shingyoji, Masato; Sekine, Ikuo; Kobayashi, Hiroshi; Tada, Yuji; Tatsumi, Koichiro; Hiroshima, Kenzo; Shimada, Hideaki; Tagawa, Masatoshi

2015-01-01

Improvement of transduction and augmentation of cytotoxicity are crucial for adenoviruses (Ad)-mediated gene therapy for cancer. Down-regulated expression of type 5 Ad (Ad5) receptors on human tumors hampered Ad-mediated transduction. Furthermore, a role of the p53 pathways in cytotoxicity mediated by replication-competent Ad remained uncharacterized. We constructed replication-competent Ad5 of which the E1 region genes were activated by a transcriptional regulatory region of the midkine or the survivin gene, which is expressed preferentially in human tumors. We also prepared replication-competent Ad5 which were regulated by the same region but had a fiber-knob region derived from serotype 35 (AdF35). We examined the cytotoxicity of these Ad and a possible combinatory use of the replication-competent AdF35 and Ad5 expressing the wild-type p53 gene (Ad5/p53) in esophageal carcinoma cells. Expression levels of molecules involved in cell death, anti-tumor effects in vivo and production of viral progenies were also investigated. Replication-competent AdF35 in general achieved greater cytotoxic effects to esophageal carcinoma cells than the corresponding replication-competent Ad5. Infection with the AdF35 induced cleavages of caspases and increased sub-G1 fractions, but did not activate the autophagy pathway. Transduction with Ad5/p53 in combination with the replication-competent AdF35 further enhanced the cytotoxicity in a synergistic manner. We also demonstrated the combinatory effects in an animal model. Transduction with Ad5/p53 however suppressed production of replication-competent AdF35 progenies, but the combination augmented Ad5/p53-mediated p53 expression levels and the downstream pathways. Combination of replication-competent AdF35 and Ad5/p53 achieved synergistic cytotoxicity due to enhanced p53-mediated apoptotic pathways. The online version of this article (doi:10.1186/s12885-015-1482-8) contains supplementary material, which is available to authorized

Gene expression underlying enhanced, steroid-dependent auditory sensitivity of hair cell epithelium in a vocal fish.

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Fergus, Daniel J; Feng, Ni Y; Bass, Andrew H

2015-10-14

Successful animal communication depends on a receiver's ability to detect a sender's signal. Exemplars of adaptive sender-receiver coupling include acoustic communication, often important in the context of seasonal reproduction. During the reproductive summer season, both male and female midshipman fish (Porichthys notatus) exhibit similar increases in the steroid-dependent frequency sensitivity of the saccule, the main auditory division of the inner ear. This form of auditory plasticity enhances detection of the higher frequency components of the multi-harmonic, long-duration advertisement calls produced repetitively by males during summer nights of peak vocal and spawning activity. The molecular basis of this seasonal auditory plasticity has not been fully resolved. Here, we utilize an unbiased transcriptomic RNA sequencing approach to identify differentially expressed transcripts within the saccule's hair cell epithelium of reproductive summer and non-reproductive winter fish. We assembled 74,027 unique transcripts from our saccular epithelial sequence reads. Of these, 6.4 % and 3.0 % were upregulated in the reproductive and non-reproductive saccular epithelium, respectively. Gene ontology (GO) term enrichment analyses of the differentially expressed transcripts showed that the reproductive saccular epithelium was transcriptionally, translationally, and metabolically more active than the non-reproductive epithelium. Furthermore, the expression of a specific suite of candidate genes, including ion channels and components of steroid-signaling pathways, was upregulated in the reproductive compared to the non-reproductive saccular epithelium. We found reported auditory functions for 14 candidate genes upregulated in the reproductive midshipman saccular epithelium, 8 of which are enriched in mouse hair cells, validating their hair cell-specific functions across vertebrates. We identified a suite of differentially expressed genes belonging to neurotransmission and

Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch

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Arumugam, Aadithya; Weng, Zhiping; Chaudhary, Sandeep C.; Afaq, Farrukh [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Elmets, Craig A. [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

2014-08-29

Highlights: • Targeting ODC to hair follicle augments skin carcinogenesis and invasive SCCs. • Hair follicle ODC expands stem cell compartment carrying CD34{sup +}/K15{sup +}/p63{sup +} keratinocytes. • Negatively regulated Notch1 is associated with expansion of stem cell compartment. - Abstract: Over-expression of ornithine decarboxylase (ODC) is known to be involved in the epidermal carcinogenesis. However, the mechanism by which it enhances skin carcinogenesis remains undefined. Recently, role of stem cells localized in various epidermal compartments has been shown in the pathogenesis of skin cancer. To direct ODC expression in distinct epidermal compartments, we have developed keratin 6 (K6)-ODC/SKH-1 and keratin 14 (K14)-ODC/SKH-1 mice and employed them to investigate the role of ODC directed to these epidermal compartments on UVB-induced carcinogenesis. K6-driven ODC over-expression directed to outer root sheath (ORS) of hair follicle was more effective in augmenting tumorigenesis as compared to mice where K14-driven ODC expression was directed to inter-follicular epidermal keratinocytes. Chronically UVB-irradiated K6-ODC/SKH-1 developed 15 ± 2.5 tumors/mouse whereas K14-ODC/SKH-1 developed only 6.8 ± 1.5 tumors/mouse. K6-ODC/SKH-1 showed augmented UVB-induced proliferation and much higher pro-inflammatory responses than K14-ODC/SKH-1 mice. Tumors induced in K6-ODC/SKH-1 were rapidly growing, invasive and ulcerative squamous cell carcinoma (SCC) showing decreased expression of epidermal polarity marker E-cadherin and enhanced mesenchymal marker, fibronectin. Interestingly, the number of CD34/CK15/p63 positive stem-like cells was significantly higher in chronically UVB-irradiated K6-ODC/SKH-1 as compared to K14-ODC/SKH-1 mice. Reduced Notch1 expression was correlated with the expansion of stem cell compartment in these animals. However, other signaling pathways such as DNA damage response or mTOR signaling pathways were not significantly different in

Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch

International Nuclear Information System (INIS)

Arumugam, Aadithya; Weng, Zhiping; Chaudhary, Sandeep C.; Afaq, Farrukh; Elmets, Craig A.; Athar, Mohammad

2014-01-01

Highlights: • Targeting ODC to hair follicle augments skin carcinogenesis and invasive SCCs. • Hair follicle ODC expands stem cell compartment carrying CD34 + /K15 + /p63 + keratinocytes. • Negatively regulated Notch1 is associated with expansion of stem cell compartment. - Abstract: Over-expression of ornithine decarboxylase (ODC) is known to be involved in the epidermal carcinogenesis. However, the mechanism by which it enhances skin carcinogenesis remains undefined. Recently, role of stem cells localized in various epidermal compartments has been shown in the pathogenesis of skin cancer. To direct ODC expression in distinct epidermal compartments, we have developed keratin 6 (K6)-ODC/SKH-1 and keratin 14 (K14)-ODC/SKH-1 mice and employed them to investigate the role of ODC directed to these epidermal compartments on UVB-induced carcinogenesis. K6-driven ODC over-expression directed to outer root sheath (ORS) of hair follicle was more effective in augmenting tumorigenesis as compared to mice where K14-driven ODC expression was directed to inter-follicular epidermal keratinocytes. Chronically UVB-irradiated K6-ODC/SKH-1 developed 15 ± 2.5 tumors/mouse whereas K14-ODC/SKH-1 developed only 6.8 ± 1.5 tumors/mouse. K6-ODC/SKH-1 showed augmented UVB-induced proliferation and much higher pro-inflammatory responses than K14-ODC/SKH-1 mice. Tumors induced in K6-ODC/SKH-1 were rapidly growing, invasive and ulcerative squamous cell carcinoma (SCC) showing decreased expression of epidermal polarity marker E-cadherin and enhanced mesenchymal marker, fibronectin. Interestingly, the number of CD34/CK15/p63 positive stem-like cells was significantly higher in chronically UVB-irradiated K6-ODC/SKH-1 as compared to K14-ODC/SKH-1 mice. Reduced Notch1 expression was correlated with the expansion of stem cell compartment in these animals. However, other signaling pathways such as DNA damage response or mTOR signaling pathways were not significantly different in tumors induced

KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

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Park Jung-Jin

2012-03-01

Full Text Available Abstract Background KAI1 was initially identified as a metastasis-suppressor gene in prostate cancer. It is a member of the tetraspan transmembrane superfamily (TM4SF of membrane glycoproteins. As part of a tetraspanin-enriched microdomain (TEM, KAI1 inhibits tumor metastasis by negative regulation of Src. However, the underlying regulatory mechanism has not yet been fully elucidated. CUB-domain-containing protein 1 (CDCP1, which was previously known as tetraspanin-interacting protein in TEM, promoted metastasis via enhancement of Src activity. To better understand how KAI1 is involved in the negative regulation of Src, we here examined the function of KAI1 in CDCP1-mediated Src kinase activation and the consequences of this process, focusing on HIF-1 α and VEGF expression. Methods We used the human prostate cancer cell line PC3 which was devoid of KAI1 expression. Vector-transfected cells (PC3-GFP clone #8 and KAI1-expressing PC3 clones (PC3-KAI1 clone #5 and #6 were picked after stable transfection with KAI1 cDNA and selection in 800 μg/ml G418. Protein levels were assessed by immunoblotting and VEGF reporter gene activity was measured by assaying luciferase activitiy. We followed tumor growth in vivo and immunohistochemistry was performed for detection of HIF-1, CDCP1, and VHL protein level. Results We demonstrated that Hypoxia-inducible factor 1α (HIF-1α and VEGF expression were significantly inhibited by restoration of KAI1 in PC3 cells. In response to KAI1 expression, CDCP1-enhanced Src activation was down-regulated and the level of von Hippel-Lindau (VHL protein was significantly increased. In an in vivo xenograft model, KAI1 inhibited the expression of CDCP1 and HIF-1α. Conclusions These novel observations may indicate that KAI1 exerts profound metastasis-suppressor activity in the tumor malignancy process via inhibition of CDCP1-mediated Src activation, followed by VHL-induced HIF-1α degradation and, ultimately, decreased VEGF

Delayed expression of enhanced reactivation and decreased mutagenesis of UV-irradiated adenovirus in UV-irradiated ataxia telangiectasia fibroblasts

International Nuclear Information System (INIS)

Bennett, C.B.; Rainbow, A.J.

1988-01-01

In this study the authors examined UV-enhanced reactivation (UVER) and UV-enhanced mutagenesis (UVEM) of UV-irradiated adenovirus in AT fibroblasts. UVER factors for Ad V antigen expression were significantly less than normal in AT strains tested when infection occurred immediately after UV-irradiation of cells. However, UVER factors were >1 and similar to those found for normal strains when cells were infected 24 h after UV-irradiation, indicating delay in the expression of UVER for Ad V antigen in AT cells. UV-irradiation of both normal and AT cells 24 h prior to infection also resulted in a significant increase in progeny survival for UV-irradiated Ad. In normal cells, this progeny UVER was concomitant with a significant increase in the mutation frequency for UV-irradiated virus (increase in targeted mutagenesis) suggesting existence of an inducible error-prone DNA repair mode in normal human cells. In contrast, pre-UV-irradiation of AT cells resulted in a significant decrease in the mutation frequency for UV-irradiated virus. (author)

Local gene expression changes after UV-irradiation of human skin.

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Benjamin Weinkauf

Full Text Available UV-irradiation is a well-known translational pain model inducing local inflammation and primary hyperalgesia. The mediators and receptor proteins specifically contributing to mechanical or heat hyperalgesia are still unclear. Therefore, we irradiated buttock skin of humans (n = 16 with 5-fold MED of UV-C and assessed the time course of hyperalgesia and axon reflex erythema. In parallel, we took skin biopsies at 3, 6 and 24 h after UVC irradiation and assessed gene expression levels (RT-PCR of neurotrophins (e.g. NGF, BDNF, GDNF, ion channels (e.g. NaV1.7, TRPV1, inflammatory mediators (e.g. CCL-2, CCL-3 and enzymes (e.g. PGES, COX2. Hyperalgesia to mechanical impact (12 m/s and heat (48 °C stimuli was significant at 6 h (p<0.05 and p<0.01 and 24 h (p<0.005 and p<0.01 after irradiation. Axon reflex erythema upon mechanical and thermal stimuli was significantly increased 3 h after irradiation and particularly strong at 6 h. A significant modulation of 9 genes was found post UV-C irradiation, including NGF (3, 6, 24 h, TrkA (6, 24 h, artemin, bradykinin-1 receptor, COX-2, CCL-2 and CCL-3 (3 and 6 h each. A significant down-regulation was observed for TRPV1 and iNOS (6, 24 h. Individual one-to-one correlation analysis of hyperalgesia and gene expression revealed that changes of Nav1.7 (SCN9A mRNA levels at 6 and 24 h correlated to the intensity of mechanical hyperalgesia recorded at 24 h post UV-irradiation (Pearson r: 0.57, p<0.04 and r: 0.82, p<0.001. Expression of COX-2 and mPGES at 6 h correlated to the intensity of heat-induced erythema 24 h post UV (r: 0.57, p<0.05 for COX-2 and r: 0.83, p<0.001 for PGES. The individual correlation analyses of functional readouts (erythema and pain response with local expression changes provided evidence for a potential role of Nav1.7 in mechanical hyperalgesia.

Enhanced motivation to alcohol in transgenic mice expressing human α-synuclein.

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Rotermund, Carola; Reolon, Gustavo K; Leixner, Sarah; Boden, Cindy; Bilbao, Ainhoa; Kahle, Philipp J

2017-11-01

α-Synuclein (αSYN) is the neuropathological hallmark protein of Parkinson's disease (PD) and related neurodegenerative disorders. Moreover, the gene encoding αSYN (SNCA) is a major genetic contributor to PD. Interestingly, independent genome-wide association studies also identified SNCA as the most important candidate gene for alcoholism. Furthermore, single-nucleotide-polymorphisms have been associated with alcohol-craving behavior and alcohol-craving patients showed augmented αSYN expression in blood. To investigate the effect of αSYN on the addictive properties of chronic alcohol use, we examined consumption, motivation, and seeking responses induced by environmental stimuli and relapse behavior in transgenic mice expressing the human mutant [A30P]αSYN throughout the brain. The primary reinforcing effects of alcohol under operant self-administration conditions were increased, while consumption and the alcohol deprivation effect were not altered in the transgenic mice. The same mice were subjected to immunohistochemical measurements of immediate-early gene inductions in brain regions involved in addiction-related behaviors. Acute ethanol injection enhanced immunostaining for the phosphorylated form of cAMP response element binding protein in both amygdala and nucleus accumbens of αSYN transgenic mice, while in wild-type mice no effect was visible. However, at the same time, levels of cFos remain unchanged in both genotypes. These results provide experimental confirmation of SNCA as a candidate gene for alcoholism in addition to its known link to PD. © 2017 International Society for Neurochemistry.

Kokumi Substances, Enhancers of Basic Tastes, Induce Responses in Calcium-Sensing Receptor Expressing Taste Cells

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Maruyama, Yutaka; Yasuda, Reiko; Kuroda, Motonaka; Eto, Yuzuru

2012-01-01

Recently, we reported that calcium-sensing receptor (CaSR) is a receptor for kokumi substances, which enhance the intensities of salty, sweet and umami tastes. Furthermore, we found that several γ-glutamyl peptides, which are CaSR agonists, are kokumi substances. In this study, we elucidated the receptor cells for kokumi substances, and their physiological properties. For this purpose, we used Calcium Green-1 loaded mouse taste cells in lingual tissue slices and confocal microscopy. Kokumi substances, applied focally around taste pores, induced an increase in the intracellular Ca2+ concentration ([Ca2+]i) in a subset of taste cells. These responses were inhibited by pretreatment with the CaSR inhibitor, NPS2143. However, the kokumi substance-induced responses did not require extracellular Ca2+. CaSR-expressing taste cells are a different subset of cells from the T1R3-expressing umami or sweet taste receptor cells. These observations indicate that CaSR-expressing taste cells are the primary detectors of kokumi substances, and that they are an independent population from the influenced basic taste receptor cells, at least in the case of sweet and umami. PMID:22511946

EGCG Enhances Cisplatin Sensitivity by Regulating Expression of the Copper and Cisplatin Influx Transporter CTR1 in Ovary Cancer.

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Xuemin Wang

Full Text Available Cisplatin is one of the first-line platinum-based chemotherapeutic agents for treatment of many types of cancer, including ovary cancer. CTR1 (copper transporter 1, a transmembrane solute carrier transporter, has previously been shown to increase the cellular uptake and sensitivity of cisplatin. It is hypothesized that increased CTR1 expression would enhance the sensitivity of cancer cells to cisplatin (cDDP. The present study demonstrates for the first time that (--epigallocatechin-3-gallate (EGCG, a major polyphenol from green tea, can enhance CTR1 mRNA and protein expression in ovarian cancer cells and xenograft mice. EGCG inhibits the rapid degradation of CTR1 induced by cDDP. The combination of EGCG and cDDP increases the accumulation of cDDP and DNA-Pt adducts, and subsequently enhances the sensitivity of ovarian cancer SKOV3 and OVCAR3 cells to the chemotherapeutic agent. In the OVCAR3 ovarian cancer xenograft nude mice model, the combination of the lower concentration of cDDP and EGCG strongly repressed the tumor growth and exhibited protective effect on the nephrotoxicity induced by cisplatin. Overall, these findings uncover a novel chemotherapy mechanism of EGCG as an adjuvant for the treatment of ovarian cancer.

MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression.

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Xu, Meixiang; Cross, Courtney E; Speidel, Jordan T; Abdel-Rahman, Sherif Z

2016-10-01

The O 6 -methylguanine-DNA methyltransferase (MGMT) protein removes O 6 -alkyl-guanine adducts from DNA. MGMT expression can thus alter the sensitivity of cells and tissues to environmental and chemotherapeutic alkylating agents. Previously, we defined the haplotype structure encompassing single nucleotide polymorphisms (SNPs) in the MGMT promoter/enhancer (P/E) region and found that haplotypes, rather than individual SNPs, alter MGMT promoter activity. The exact mechanism(s) by which these haplotypes exert their effect on MGMT promoter activity is currently unknown, but we noted that many of the SNPs comprising the MGMT P/E haplotypes are located within or in close proximity to putative transcription factor binding sites. Thus, these haplotypes could potentially affect transcription factor binding and, subsequently, alter MGMT promoter activity. In this study, we test the hypothesis that MGMT P/E haplotypes affect MGMT promoter activity by altering transcription factor (TF) binding to the P/E region. We used a promoter binding TF profiling array and a reporter assay to evaluate the effect of different P/E haplotypes on TF binding and MGMT expression, respectively. Our data revealed a significant difference in TF binding profiles between the different haplotypes evaluated. We identified TFs that consistently showed significant haplotype-dependent binding alterations (p ≤ 0.01) and revealed their role in regulating MGMT expression using siRNAs and a dual-luciferase reporter assay system. The data generated support our hypothesis that promoter haplotypes alter the binding of TFs to the MGMT P/E and, subsequently, affect their regulatory function on MGMT promoter activity and expression level.

Momordica charantia ointment accelerates diabetic wound healing and enhances transforming growth factor-β expression.

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Hussan, F; Teoh, S Lin; Muhamad, N; Mazlan, M; Latiff, A A

2014-08-01

Transforming growth factor-β (TGF-β) plays an important role in wound healing. Delayed wound healing is a consequence of diabetes, leading to high morbidity and poor quality of life. Momordica charantia (MC) fruit possesses anti-diabetic and wound healing properties. This study aimed to explore the changes in TGF-β expression in diabetic wounds treated with topical MC fruit extract. Fifty-six male Sprague-Dawley rats were divided into a normal control group and five diabetic groups of ten rats each. Intravenous streptozotocin (50mg/kg) was given to induce diabetes in the diabetic groups. Full thickness excision wounds were created on the thoracodorsal region of the animals, and these wounds were then treated with vehicle, MC powder, MC ointment and povidone ointment or ointment base for ten days. Wound healing was determined by the rate of wound closure, total protein content and TGF-β expression in the wounds, and histological observation. Diabetic groups showed delayed wound closure rates compared to the control group. The wound closure rate in the MC ointment group was significantly faster than that of the untreated diabetic group (p<0.05). The MC ointment group also showed intense TGF-β expression and a high level of total protein content. MC ointment has a promising potential for use as an alternative topical medication for diabetic wounds. This work has shown that it accelerates wound healing in diabetic rats, and it is suggested here that this occurs by enhancing TGF-β expression. Further work is recommended to explore this effect.

The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development.

Science.gov (United States)

Huang, Wen; Deng, Yun; Dong, Wei; Yuan, Wuzhou; Wan, Yongqi; Mo, Xiaoyan; Li, Yongqing; Wang, Zequn; Wang, Yuequn; Ocorr, Karen; Zhang, Bo; Lin, Shuo; Wu, Xiushan

2011-02-01

In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.

Ectopically expressing MdPIP1;3, an aquaporin gene, increased fruit size and enhanced drought tolerance of transgenic tomatoes.

Science.gov (United States)

Wang, Lin; Li, Qing-Tian; Lei, Qiong; Feng, Chao; Zheng, Xiaodong; Zhou, Fangfang; Li, Lingzi; Liu, Xuan; Wang, Zhi; Kong, Jin

2017-12-19

Water deficit severely reduces apple growth and production, is detrimental to fruit quality and size. This problem is exacerbated as global warming is implicated in producing more severe drought stress. Thus water-efficiency has becomes the major target for apple breeding. A desired apple tree can absorb and transport water efficiently, which not only confers improved drought tolerance, but also guarantees fruit size for higher income returns. Aquaporins, as water channels, control water transportation across membranes and can regulate water flow by changing their amount and activity. The exploration of molecular mechanism of water efficiency and the gene wealth will pave a way for molecular breeding of drought tolerant apple tree. In the current study, we screened out a drought inducible aquaporin gene MdPIP1;3, which specifically enhanced its expression during fruit expansion in 'Fuji' apple (Malus domestica Borkh. cv. Red Fuji). It localized on plasma membranes and belonged to PIP1 subfamily. The tolerance to drought stress enhanced in transgenic tomato plants ectopically expressing MdPIP1;3, showing that the rate of losing water in isolated transgenic leaves was slower than wild type, and stomata of transgenic plants closed sensitively to respond to drought compared with wild type. Besides, length and diameter of transgenic tomato fruits increased faster than wild type, and in final, fruit sizes and fresh weights of transgenic tomatoes were bigger than wild type. Specially, in cell levels, fruit cell size from transgenic tomatoes was larger than wild type, showing that cell number per mm 2 in transgenic fruits was less than wild type. Altogether, ectopically expressing MdPIP1;3 enhanced drought tolerance of transgenic tomatoes partially via reduced water loss controlled by stomata closure in leaves. In addition, the transgenic tomato fruits are larger and heavier with larger cells via more efficient water transportation across membranes. Our research will

Tyk2 expression and its signaling enhances the invasiveness of prostate cancer cells

International Nuclear Information System (INIS)

Ide, Hisamitsu; Nakagawa, Takashi; Terado, Yuichi; Kamiyama, Yutaka; Muto, Satoru; Horie, Shigeo

2008-01-01

Protein tyrosine kinase plays a central role in the proliferation and differentiation of various types of cells. One of these protein kinases, Tyk2, a member of the Jak family kinases, is known to play important roles in receptor signal transduction by interferons, interleukins, growth factors, and other hormones. In the present study, we investigated Tyk2 expression and its role in the growth and invasiveness of human prostate cancer cells. We used a small interfering RNA targeting Tyk2 and an inhibitor of Tyk2, tyrphostin A1, to suppress the expression and signaling of Tyk2 in prostate cancer cells. We detected mRNAs for Jak family kinases in prostate cancer cell lines by RT-PCR and Tyk2 protein in human prostate cancer specimens by immunohistochemistry. Inhibition of Tyk2 signaling resulted in attenuation of the urokinase-type plasminogen activator-enhanced invasiveness of prostate cancer cells in vitro without affecting the cellular growth rate. These results suggest that Tyk2 signaling in prostate cancer cells facilitate invasion of these cells, and interference with this signaling may be a potential therapeutic pathway

Inhibition of TRPA1 channel activity in sensory neurons by the glial cell line-derived neurotrophic factor family member, artemin

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Wang Shenglan

2011-05-01

Full Text Available Abstract Background The transient receptor potential (TRP channel subtype A1 (TRPA1 is known to be expressed on sensory neurons and respond to changes in temperature, pH and local application of certain noxious chemicals such as allyl isothiocyanate (AITC. Artemin is a neuronal survival and differentiation factor and belongs to the glial cell line-derived neurotrophic factor (GDNF family. Both TRPA1 and artemin have been reported to be involved in pathological pain initiation and maintenance. In the present study, using whole-cell patch clamp recording technique, in situ hybridization and behavioral analyses, we examined the functional interaction between TRPA1 and artemin. Results We found that 85.8 ± 1.9% of TRPA1-expressing neurons also expressed GDNF family receptor alpha 3 (GFR α3, and 87.5 ± 4.1% of GFRα3-expressing neurons were TRPA1-positive. In whole-cell patch clamp analysis, a short-term treatment of 100 ng/ml artemin significantly suppressed the AITC-induced TRPA1 currents. A concentration-response curve of AITC resulting from the effect of artemin showed that this inhibition did not change EC50 but did lower the AITC-induced maximum response. In addition, pre-treatment of artemin significantly suppressed the number of paw lifts induced by intraplantar injection of AITC, as well as the formalin-induced pain behaviors. Conclusions These findings that a short-term application of artemin inhibits the TRPA1 channel's activity and the sequential pain behaviors suggest a role of artemin in regulation of sensory neurons.

Relationship of preoperative gastric cancer CT enhancement ratio and perfusion parameters with serum tumor marker levels and proliferation molecule expression in tumor lesions

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Yong-Hong Wang

2017-06-01

Full Text Available Objective: To study the relationship of preoperative gastric cancer CT enhancement ratio and perfusion parameters with serum tumor marker levels and proliferation molecule expression in tumor lesions. Methods: A total of 68 patients with gastric cancer treated in the Second Hospital of Yulin City between May 2012 and May 2016 were chosen as observation group and sub-divided into early and middle gastric cancer group (n=41 and advanced gastric cancer group (n=27 according to the tumor stage; 50 patients diagnosed with benign gastric diseases in our hospital during the same period were selected as benign gastric lesion group. CT enhancement rate and perfusion parameters of three groups of patients were detected by CT scan, serum tumor marker levels were evacuated by enzyme-linked immunosorbent assay (ELISA, and the proliferation gene mRNA expression levels were detected by RTPCR method. Results: CER, AF, BV and CL levels of advanced gastric cancer group were higher than those of early and middle gastric cancer group and benign gastric lesion group; serum CA72-4, CA19-9, CA125 and CEA contents of advanced gastric cancer group were higher than those of early and middle gastric cancer group and benign gastric lesion group; CADM1, miRNA-34a and Cystatin M mRNA expression in tissue of advanced gastric cancer group were lower than those of early and middle gastric cancer group and benign gastric lesion group while Survivin and I2PP2A mRNA expression were higher than those of early and middle gastric cancer group and benign gastric lesion group. The Pearson test showed that the CT enhancement rate and perfusion parameters in patients with gastric cancer are directly correlated with the serum tumor marker levels and the proliferation gene expression in tumor lesions. Conclusion: Preoperative gastric cancer CT enhancement rate and perfusion parameters are directly related to the tumor malignancy, and can be used as a reliable method for the long-term tumor

Transdifferentiation of brain-derived neurotrophic factor (BDNF)-secreting mesenchymal stem cells significantly enhance BDNF secretion and Schwann cell marker proteins.

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Bierlein De la Rosa, Metzere; Sharma, Anup D; Mallapragada, Surya K; Sakaguchi, Donald S

2017-11-01

The use of genetically modified mesenchymal stem cells (MSCs) is a rapidly growing area of research targeting delivery of therapeutic factors for neuro-repair. Cells can be programmed to hypersecrete various growth/trophic factors such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF) to promote regenerative neurite outgrowth. In addition to genetic modifications, MSCs can be subjected to transdifferentiation protocols to generate neural cell types to physically and biologically support nerve regeneration. In this study, we have taken a novel approach by combining these two unique strategies and evaluated the impact of transdifferentiating genetically modified MSCs into a Schwann cell-like phenotype. After 8 days in transdifferentiation media, approximately 30-50% of transdifferentiated BDNF-secreting cells immunolabeled for Schwann cell markers such as S100β, S100, and p75 NTR . An enhancement was observed 20 days after inducing transdifferentiation with minimal decreases in expression levels. BDNF production was quantified by ELISA, and its biological activity tested via the PC12-TrkB cell assay. Importantly, the bioactivity of secreted BDNF was verified by the increased neurite outgrowth of PC12-TrkB cells. These findings demonstrate that not only is BDNF actively secreted by the transdifferentiated BDNF-MSCs, but also that it has the capacity to promote neurite sprouting and regeneration. Given the fact that BDNF production remained stable for over 20 days, we believe that these cells have the capacity to produce sustainable, effective, BDNF concentrations over prolonged time periods and should be tested within an in vivo system for future experiments. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Transgenic expression of soluble human CD5 enhances experimentally-induced autoimmune and anti-tumoral immune responses.

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Rafael Fenutría

Full Text Available CD5 is a lymphoid-specific transmembrane glycoprotein constitutively expressed on thymocytes and mature T and B1a lymphocytes. Current data support the view that CD5 is a negative regulator of antigen-specific receptor-mediated signaling in these cells, and that this would likely be achieved through interaction with CD5 ligand/s (CD5L of still undefined nature expressed on immune or accessory cells. To determine the functional consequence of loss of CD5/CD5L interaction in vivo, a new transgenic mouse line was generated (shCD5EμTg, expressing a circulating soluble form of human CD5 (shCD5 as a decoy to impair membrane-bound CD5 function. These shCD5EμTg mice showed an enhanced response to autologous antigens, as deduced from the presentation of more severe forms of experimentally inducible autoimmune disease (collagen-induced arthritis, CIA; and experimental autoimmune encephalitis, EAE, as well as an increased anti-tumoral response in non-orthotopic cancer models (B16 melanoma. This enhancement of the immune response was in agreement with the finding of significantly reduced proportions of spleen and lymph node Treg cells (CD4+CD25+FoxP3+, and of peritoneal IL-10-producing and CD5+ B cells, as well as an increased proportion of spleen NKT cells in shCD5EμTg mice. Similar changes in lymphocyte subpopulations were observed in wild-type mice following repeated administration of exogenous recombinant shCD5 protein. These data reveal the relevant role played by CD5/CD5L interactions on the homeostasis of some functionally relevant lymphocyte subpopulations and the modulation of immune responses to autologous antigens.

Heterologous Expression of Panax ginseng PgTIP1 Confers Enhanced Salt Tolerance of Soybean Cotyledon Hairy Roots, Composite, and Whole Plants

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Jing An

2017-07-01

Full Text Available The Panax ginseng TIP gene PgTIP1 was previously demonstrated to have high water channel activity by its heterologous expression in Xenopus laevis oocytes and in yeast; it also plays a significant role in growth of PgTIP1-transgenic Arabidopsis plants under favorable conditions and has enhanced tolerance toward salt and drought treatment. In this work, we first investigated the physiological effects of heterologous PgTIP1 expression in soybean cotyledon hairy roots or composite plants mediated by Agrobacterium rhizogenes toward enhanced salt tolerance. The PgTIP1-transgenic soybean plants mediated by the pollen tube pathway, represented by the lines N and J11, were analyzed at the physiological and molecular levels for enhanced salt tolerance. The results showed that in terms of root-specific heterologous expression, the PgTIP1-transformed soybean cotyledon hairy roots or composite plants displayed superior salt tolerance compared to the empty vector-transformed ones according to the mitigatory effects of hairy root growth reduction, drop in leaf RWC, and rise in REL under salt stress. Additionally, declines in K+ content, increases in Na+ content and Na+/K+ ratios in the hairy roots, stems, or leaves were effectively alleviated by PgTIP1-transformation, particularly the stems and leaves of composite soybean plants. At the whole plant level, PgTIP1-trasgenic soybean lines were found to possess stronger root vigor, reduced root and leaf cell membrane damage, increased SOD, POD, CAT, and APX activities, steadily increased leaf Tr, RWC, and Pn values, and smaller declines in chlorophyll and carotenoid content when exposed to salt stress compared to wild type. Moreover, the distribution patterns of Na+, K+, and Cl- in the roots, stems, and leaves of salt-stressed transgenic plants were readjusted, in that the absorbed Na+ and Cl- were mainly restricted to the roots to reduce their transport to the shoots, and the transport of root-absorbed K+ to the

L-malate enhances the gene expression of carried proteins and antioxidant enzymes in liver of aged rats.

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Zeng, X; Wu, J; Wu, Q; Zhang, J

2015-01-01

Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. To investigate the antioxidant mechanism of L-malate in the mitochondria, we analyzed the change in gene expression of two malate-aspartate shuttle (MAS)-related carried proteins (AGC, aspartate/glutamate carrier and OMC, oxoglutarate/malate carrier) in the inner mitochondrial membrane, and three antioxidant enzymes (CAT, SOD, and GSH-Px) in the mitochondria. The changes in gene expression of these proteins and enzymes were examined by real-time RT-PCR in the heart and liver of aged rats treated with L-malate. L-malate was orally administered in rats continuously for 30 days using a feeding atraumatic needle. We found that the gene expression of OMC and GSH-Px mRNA in the liver increased by 39 % and 38 %, respectively, in the 0.630 g/kg L-malate treatment group than that in the control group. The expression levels of SOD mRNA in the liver increased by 39 %, 56 %, and 78 % in the 0.105, 0.210, and 0.630 g/kg L-malate treatment groups, respectively. No difference were observed in the expression levels of AGC, OMC, CAT, SOD, and GSH-Px mRNAs in the heart of rats between the L-malate treatment and control groups. These results predicted that L-malate may increase the antioxidant capacity of mitochondria by enhancing the expression of mRNAs involved in the MAS and the antioxidant enzymes.

Enhanced production of subtilisin of Pyrococcus furiosus expressed ...

African Journals Online (AJOL)

PRECIOUS

2009-11-02

Nov 2, 2009 ... on SDS-PAGE as compared to theoretical molecular mass of 17.6 kDa. This aberrant electrophoresis mobility could be .... analyze protein expression by 12% SDS-PAGE (Laemmli, 1970). To analyze the expression of .... pellet washed with buffer containing Triton X; lane 4, refolded subtilisin. subjected to ...

Artesunate Reduces Serum Lipopolysaccharide in Cecal Ligation/Puncture Mice via Enhanced LPS Internalization by Macrophages through Increased mRNA Expression of Scavenger Receptors

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Bin Li

2014-01-01

Full Text Available Innate immunity is the first line of defense in human beings against pathogen infection; monocytes/macrophages are the primary cells of the innate immune system. Recently, macrophages/monocytes have been discovered to participate in LPS clearance, and the clearance efficiency determines the magnitude of the inflammatory response and subsequent organ injury. Previously, we reported that artesunate (AS protected sepsis mice against heat-killed E. coli challenge. Herein, we further confirmed that AS protected cecal ligation/puncture (CLP sepsis mice. Its protection on sepsis mice was related to not only reduction of pro-inflammatory cytokines and serum LPS levels but also improvement of liver function. Based on the fact that AS did not directly bind and neutralize LPS, we hypothesized that the reduction of serum LPS level might be related to enhancement of LPS internalization and subsequent detoxification. Our results showed that AS increased FITC-LPS internalization by peritoneal macrophage and liver Kupffer cell, but enhancement of LPS internalization by AS was not related to the clathrin-dependent pathway. However, AS induced mRNA expression of important scavenger receptors (SRs; SR-A and MARCO mRNA expression was upregulated, suggesting that AS enhancement of LPS internalization and inhibition of pro-inflammatory cytokines was related to changes in mRNA expression of SRs.

MHC class II super-enhancer increases surface expression of HLA-DR and HLA-DQ and affects cytokine production in autoimmune vitiligo.

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Cavalli, Giulio; Hayashi, Masahiro; Jin, Ying; Yorgov, Daniel; Santorico, Stephanie A; Holcomb, Cherie; Rastrou, Melinda; Erlich, Henry; Tengesdal, Isak W; Dagna, Lorenzo; Neff, C Preston; Palmer, Brent E; Spritz, Richard A; Dinarello, Charles A

2016-02-02

Genetic risk for autoimmunity in HLA genes is most often attributed to structural specificity resulting in presentation of self-antigens. Autoimmune vitiligo is strongly associated with the MHC class II region. Here, we fine-map vitiligo MHC class II genetic risk to three SNPs only 47 bp apart, located within a predicted super-enhancer in an intergenic region between HLA-DRB1 and HLA-DQA1, localized by a genome-wide association study of 2,853 Caucasian vitiligo patients. The super-enhancer corresponds to an expression quantitative trait locus for expression of HLA-DR and HLA-DQ RNA; we observed elevated surface expression of HLA-DR (P = 0.008) and HLA-DQ (P = 0.02) on monocytes from healthy subjects homozygous for the high-risk SNP haplotype. Unexpectedly, pathogen-stimulated peripheral blood mononuclear cells from subjects homozygous for the high-risk super-enhancer haplotype exhibited greater increase in production of IFN-γ and IL-1β than cells from subjects homozygous for the low-risk haplotype. Specifically, production of IFN-γ on stimulation of dectin-1, mannose, and Toll-like receptors with Candida albicans and Staphylococcus epidermidis was 2.5- and 2.9-fold higher in high-risk subjects than in low-risk subjects, respectively (P = 0.007 and P = 0.01). Similarly, production of IL-1β was fivefold higher in high-risk subjects than in low-risk subjects (P = 0.02). Increased production of immunostimulatory cytokines in subjects carrying the high-risk haplotype may act as an "adjuvant" during the presentation of autoantigens, tying together genetic variation in the MHC with the development of autoimmunity. This study demonstrates that for risk of autoimmune vitiligo, expression level of HLA class II molecules is as or more important than antigen specificity.

Radiation impairs perineural invasion by modulating the nerve microenvironment.

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Richard L Bakst

Full Text Available Perineural invasion (PNI by cancer cells is an ominous clinical event that is associated with increased local recurrence and poor prognosis. Although radiation therapy (RT may be delivered along the course of an invaded nerve, the mechanisms through which radiation may potentially control PNI remain undefined.An in vitro co-culture system of dorsal root ganglia (DRG and pancreatic cancer cells was used as a model of PNI. An in vivo murine sciatic nerve model was used to study how RT to nerve or cancer affects nerve invasion by cancer.Cancer cell invasion of the DRG was partially dependent on DRG secretion of glial-derived neurotrophic factor (GDNF. A single 4 Gy dose of radiation to the DRG alone, cultured with non-radiated cancer cells, significantly inhibited PNI and was associated with decreased GDNF secretion but intact DRG viability. Radiation of cancer cells alone, co-cultured with non-radiated nerves, inhibited PNI through predominantly compromised cancer cell viability. In a murine model of PNI, a single 8 Gy dose of radiation to the sciatic nerve prior to implantation of non-radiated cancer cells resulted in decreased GDNF expression, decreased PNI by imaging and histology, and preservation of sciatic nerve motor function.Radiation may impair PNI through not only direct effects on cancer cell viability, but also an independent interruption of paracrine mechanisms underlying PNI. RT modulation of the nerve microenvironment may decrease PNI, and hold significant therapeutic implications for RT dosing and field design for patients with cancers exhibiting PNI.

Chronic corticosterone exposure persistently elevates the expression of memory-related genes in the lateral amygdala and enhances the consolidation of a Pavlovian fear memory.

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Melissa S Monsey

Full Text Available Chronic exposure to stress has been widely implicated in the development of anxiety disorders, yet relatively little is known about the long-term effects of chronic stress on amygdala-dependent memory formation. Here, we examined the effects of a history of chronic exposure to the stress-associated adrenal steroid corticosterone (CORT on the consolidation of a fear memory and the expression of memory-related immediate early genes (IEGs in the lateral nucleus of the amygdala (LA. Rats received chronic exposure to CORT (50 μg/ml in their drinking water for 2 weeks and were then titrated off the CORT for an additional 6 days followed by a 2 week 'wash-out' period consisting of access to plain water. Rats were then either sacrificed to examine the expression of memory-related IEG expression in the LA or given auditory Pavlovian fear conditioning. We show that chronic exposure to CORT leads to a persistent elevation in the expression of the IEGs Arc/Arg3.1 and Egr-1 in the LA. Further, we show that rats with a history of chronic CORT exposure exhibit enhanced consolidation of a fear memory; short-term memory (STM is not affected, while long-term memory (LTM is significantly enhanced. Treatment with the selective serotonin reuptake inhibitor (SSRI fluoxetine following the chronic CORT exposure period was observed to effectively reverse both the persistent CORT-related increases in memory-related IEG expression in the LA and the CORT-related enhancement in fear memory consolidation. Our findings suggest that chronic exposure to CORT can regulate memory-related IEG expression and fear memory consolidation processes in the LA in a long-lasting manner and that treatment with fluoxetine can reverse these effects.

Chronic corticosterone exposure persistently elevates the expression of memory-related genes in the lateral amygdala and enhances the consolidation of a Pavlovian fear memory.

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Monsey, Melissa S; Boyle, Lara M; Zhang, Melinda L; Nguyen, Caroline P; Kronman, Hope G; Ota, Kristie T; Duman, Ronald S; Taylor, Jane R; Schafe, Glenn E

2014-01-01

Chronic exposure to stress has been widely implicated in the development of anxiety disorders, yet relatively little is known about the long-term effects of chronic stress on amygdala-dependent memory formation. Here, we examined the effects of a history of chronic exposure to the stress-associated adrenal steroid corticosterone (CORT) on the consolidation of a fear memory and the expression of memory-related immediate early genes (IEGs) in the lateral nucleus of the amygdala (LA). Rats received chronic exposure to CORT (50 μg/ml) in their drinking water for 2 weeks and were then titrated off the CORT for an additional 6 days followed by a 2 week 'wash-out' period consisting of access to plain water. Rats were then either sacrificed to examine the expression of memory-related IEG expression in the LA or given auditory Pavlovian fear conditioning. We show that chronic exposure to CORT leads to a persistent elevation in the expression of the IEGs Arc/Arg3.1 and Egr-1 in the LA. Further, we show that rats with a history of chronic CORT exposure exhibit enhanced consolidation of a fear memory; short-term memory (STM) is not affected, while long-term memory (LTM) is significantly enhanced. Treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine following the chronic CORT exposure period was observed to effectively reverse both the persistent CORT-related increases in memory-related IEG expression in the LA and the CORT-related enhancement in fear memory consolidation. Our findings suggest that chronic exposure to CORT can regulate memory-related IEG expression and fear memory consolidation processes in the LA in a long-lasting manner and that treatment with fluoxetine can reverse these effects.

Enhanced Expression of Interferon-γ-Induced Antigen-Processing Machinery Components in a Spontaneously Occurring Cancer

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Fulvia Cerruti

2007-11-01

Full Text Available In human tumors, changes in the surface expression and/or function of major histocompatibility complex (MHC class I antigens are frequently found and may provide malignant cells with a mechanism to escape control of the immune system. This altered human lymphocyte antigen (HLA class I phenotype can be caused by either structural alterations or dysregulation of genes encoding subunits of HLA class I antigens and/or components of the MHC class I antigen-processing machinery (APM. Herein we analyze the expression of several proteins involved in the generation of MHC class I epitopes in feline injection site sarcoma, a spontaneously occurring tumor in cats that is an informative model for the study of tumor biology in other species, including humans. Eighteen surgically removed primary fibrosarcoma lesions were analyzed, and an enhanced expression of two catalytic subunits of immunoproteasomes, PA28 and leucine aminopeptidase, was found in tumors compared to matched normal tissues. As a functional counterpart of these changes in protein levels, proteasomal activities were increased in tissue extracts from fibrosarcomas. Taken together, these results suggest that alterations in the APM system may account for reduced processing of selected tumor antigens and may potentially provide neoplastic fibroblasts with a mechanism for escape from T-cell recognition and destruction.

Development and characterization of enhanced green fluorescent protein and luciferase expressing cell line for non-destructive evaluation of tissue engineering constructs.

NARCIS (Netherlands)

Blum, J.S.; Temenoff, J.S.; Park, H.; Jansen, J.A.; Mikos, A.G.; Barry, M.A.

2004-01-01

This study investigates the utility of genetically modified cells developed for the qualitative and quantitative non-destructive evaluation of cells on biomaterials. The Fisher rat fibroblastic cell line has been genetically modified to stably express the reporter genes enhanced green fluorescence

Feeding hydroalcoholic extract powder of Lepidium meyenii (maca) enhances testicular gene expression of 3β-hydroxysteroid dehydrogenase in rats.

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Ohta, Y; Kawate, N; Inaba, T; Morii, H; Takahashi, K; Tamada, H

2017-12-01

Although feeding diets containing the extract powder of Lepidium meyenii (maca), a plant growing in Peru's Central Andes, increases serum testosterone concentration associated with enhanced ability of testosterone production by Leydig cells in male rats, changes in testicular steroidogenesis-related factors by the maca treatment are not known. This study examined the effects of maca on testicular gene expressions for luteinizing hormone receptor, steroidogenic acute regulatory protein and steroidogenic enzymes. Eight-week-old male rats were given the diets with or without (control) the maca extract powder (2%) for 6 weeks, and mRNA levels were determined by reverse transcription quantitative real-time PCR. The results showed that the testicular mRNA level of HSD3B1 (3β-hydroxysteroid dehydrogenase; 3β-HSD) increased by the treatment, whereas the levels of the other factors examined did not change. These results suggest that increased expression of 3β-HSD gene may be involved in the enhanced steroidogenic ability by the maca treatment in rat testes. © 2017 Blackwell Verlag GmbH.

Low-magnitude high-frequency vibration enhances gene expression related to callus formation, mineralization and remodeling during osteoporotic fracture healing in rats.

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Chung, Shu-Lu; Leung, Kwok-Sui; Cheung, Wing-Hoi

2014-12-01

Low magnitude high frequency vibration (LMHFV) has been shown to improve anabolic and osteogenic responses in osteoporotic intact bones and during osteoporotic fracture healing; however, the molecular response of LMHFV during osteoporotic fracture healing has not been investigated. It was hypothesized that LMHFV could enhance osteoporotic fracture healing by regulating the expression of genes related to chondrogenesis (Col-2), osteogenesis (Col-1) and remodeling (receptor activator for nuclear factor- κ B ligand (RANKL) and osteoproteger (OPG)). In this study, the effects of LMHFV on both osteoporotic and normal bone fracture healing were assessed by endpoint gene expressions, weekly radiographs, and histomorphometry at weeks 2, 4 and 8 post-treatment. LMHFV enhanced osteoporotic fracture healing by up-regulating the expression of chondrogenesis-, osteogenesis- and remodeling-related genes (Col-2 at week 4 (p=0.008), Col-1 at week 2 and 8 (p<0.001 and p=0.008) and RANKL/OPG at week 8 (p=0.045)). Osteoporotic bone had a higher response to LMHFV than normal bone and showed significantly better results as reflected by increased expression of Col-2 and Col-1 at week 2 (p<0.001 for all), larger callus width at week 2 (p=0.001), callus area at week 1 and 5(p<0.05 for all) and greater relative area of osseous tissue (p=0.002) at week 8. This study helps to understand how LMHFV regulates gene expression of callus formation, mineralization and remodeling during osteoporotic fracture healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Transgenic expression of lactoferrin imparts enhanced resistance to head blight of wheat caused by Fusarium graminearum.

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Han, Jigang; Lakshman, Dilip K; Galvez, Leny C; Mitra, Sharmila; Baenziger, Peter Stephen; Mitra, Amitava

2012-03-09

The development of plant gene transfer systems has allowed for the introgression of alien genes into plant genomes for novel disease control strategies, thus providing a mechanism for broadening the genetic resources available to plant breeders. Using the tools of plant genetic engineering, a broad-spectrum antimicrobial gene was tested for resistance against head blight caused by Fusarium graminearum Schwabe, a devastating disease of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) that reduces both grain yield and quality. A construct containing a bovine lactoferrin cDNA was used to transform wheat using an Agrobacterium-mediated DNA transfer system to express this antimicrobial protein in transgenic wheat. Transformants were analyzed by Northern and Western blots to determine lactoferrin gene expression levels and were inoculated with the head blight disease fungus F. graminearum. Transgenic wheat showed a significant reduction of disease incidence caused by F. graminearum compared to control wheat plants. The level of resistance in the highly susceptible wheat cultivar Bobwhite was significantly higher in transgenic plants compared to control Bobwhite and two untransformed commercial wheat cultivars, susceptible Wheaton and tolerant ND 2710. Quantification of the expressed lactoferrin protein by ELISA in transgenic wheat indicated a positive correlation between the lactoferrin gene expression levels and the levels of disease resistance. Introgression of the lactoferrin gene into elite commercial wheat, barley and other susceptible cereals may enhance resistance to F. graminearum.

Identification of an enhancer element of class Pi glutathione S-transferase gene required for expression by a co-planar polychlorinated biphenyl.

Science.gov (United States)

Matsumoto, M; Imagawa, M; Aoki, Y

1999-01-01

3,3',4,4',5-Pentachlorobiphenyl (PenCB), one of the most toxic co-planar polychlorinated biphenyl congeners, specifically induces class Pi glutathione S-transferase (GSTP1) as well as cytochrome P-450 1A1 in primary cultured rat liver parenchymal cells [Aoki, Matsumoto and Suzuki (1993) FEBS Lett. 333, 114-118]. However, the 5'-flanking sequence of the GSTP1 gene does not contain a xenobiotic responsive element, to which arylhydrocarbon receptor binds. Using a chloramphenicol acetyltransferase assay we demonstrate here that the enhancer termed GSTP1 enhancer I (GPEI) is necessary for the stimulation by PenCB of GSTP1 gene expression in primary cultured rat liver parenchymal cells. GPEI is already known to contain a dyad of PMA responsive element-like elements oriented palindromically. It is suggested that a novel signal transduction pathway activated by PenCB contributes to the stimulation of GSTP1 expression. PMID:10051428

S100B protein, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in human milk.

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Ruisong Li

Full Text Available Human milk contains a wide variety of nutrients that contribute to the fulfillment of its functions, which include the regulation of newborn development. However, few studies have investigated the concentrations of S100B protein, brain-derived neurotrophic factor (BDNF, and glial cell line-derived neurotrophic factor (GDNF in human milk. The associations of the concentrations of S100B protein, BDNF, and GDNF with maternal factors are not well explored.To investigate the concentrations of S100B protein, BDNF, and GDNF in human milk and characterize the maternal factors associated with their levels in human milk, human milk samples were collected at days 3, 10, 30, and 90 after parturition. Levels of S100B protein, BDNF, and GDNF, and their mRNAs in the samples were detected. Then, these concentrations were compared with lactation and other maternal factors. S100B protein levels in human milk samples collected at 3, 10, 30, and 90 d after parturition were 1249.79±398.10, 1345.05±539.16, 1481.83±573.30, and 1414.39±621.31 ng/L, respectively. On the other hand, the BDNF concentrations in human milk samples were 10.99±4.55, 13.01±5.88, 13.35±6.43, and 2.83±5.47 µg/L, while those of GDNF were 10.90±1.65, 11.38±1., 11.29±3.10, and 11.40±2.21 g/L for the same time periods. Maternal post-pregnancy body mass index was positively associated with S100B levels in human milk (r = 0.335, P = 0.030<0.05. In addition, there was a significant correlation between the levels of S100B protein and BDNF (z = 2.09, P = 0.037<0.05. Delivery modes were negatively associated with the concentration of GDNF in human milk.S100B protein, BDNF, and GDNF are present in all samples of human milk, and they may be responsible for the long term effects of breast feeding.

Long-term insulin-like growth factor-I expression in skeletal muscles attenuates the enhanced in vitro proliferation ability of the resident satellite cells in transgenic mice

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Chakravarthy, M. V.; Fiorotto, M. L.; Schwartz, R. J.; Booth, F. W.

2001-01-01

Insulin-like growth factor-I (IGF-I) overexpression for 1-month in mouse skeletal muscle increases satellite cell proliferation potential. However, it is unknown whether this beneficial enhancement by IGF-I expression would persist over a longer-term duration in aged mice. This is an important issue to address if a prolonged course of IGF-I is to be used clinically in muscle-wasting conditions where satellite cells may become limiting. Using the IGF-I transgenic (IGF-I Tg) mouse that selectively expresses the IGF-I transgene in striated muscles, we found that 18-months of continuous IGF-I overexpression led to a loss in the enhanced in vitro proliferative capacity of satellite cells from Tg skeletal muscles. Also 18-month-old IGF-I Tg satellite cells lost the enhanced BrdU incorporation, greater pRb and Akt phosphorylations, and decreased p27(Kip1) levels initially observed in cells from 1-month-old IGF-I Tg mice. The levels of those biochemical markers reverted to similar values seen in the 18-months WT littermates. These findings, therefore, suggest that there is no further beneficial effect on enhancing satellite cell proliferation ability with persistent long-term expression of IGF-I in skeletal muscles of these transgenic mice.

Interleukin-3 enhances the migration of human mesenchymal stem cells by regulating expression of CXCR4.

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Barhanpurkar-Naik, Amruta; Mhaske, Suhas T; Pote, Satish T; Singh, Kanupriya; Wani, Mohan R

2017-07-14

Mesenchymal stem cells (MSCs) represent an important source for cell therapy in regenerative medicine. MSCs have shown promising results for repair of damaged tissues in various degenerative diseases in animal models and also in human clinical trials. However, little is known about the factors that could enhance the migration and tissue-specific engraftment of exogenously infused MSCs for successful regenerative cell therapy. Previously, we have reported that interleukin-3 (IL-3) prevents bone and cartilage damage in animal models of rheumatoid arthritis and osteoarthritis. Also, IL-3 promotes the differentiation of human MSCs into functional osteoblasts and increases their in-vivo bone regenerative potential in immunocompromised mice. However, the role of IL-3 in migration of MSCs is not yet known. In the present study, we investigated the role of IL-3 in migration of human MSCs under both in-vitro and in-vivo conditions. MSCs isolated from human bone marrow, adipose and gingival tissues were used for in-vitro cell migration, motility and wound healing assays in the presence or absence of IL-3. The effect of IL-3 preconditioning on expression of chemokine receptors and integrins was examined by flow cytometry and real-time PCR. The in-vivo migration of IL-3-preconditioned MSCs was investigated using a subcutaneous matrigel-releasing stromal cell-derived factor-1 alpha (SDF-1α) model in immunocompromised mice. We observed that human MSCs isolated from all three sources express IL-3 receptor-α (IL-3Rα) both at gene and protein levels. IL-3 significantly enhances in-vitro migration, motility and wound healing abilities of MSCs. Moreover, IL-3 preconditioning upregulates expression of chemokine (C-X-C motif) receptor 4 (CXCR4) on MSCs, which leads to increased migration of cells towards SDF-1α. Furthermore, CXCR4 antagonist AMD3100 decreases the migration of IL-3-treated MSCs towards SDF-1α. Importantly, IL-3 also induces in-vivo migration of MSCs towards

Protection against high-fat diet-induced obesity in Helz2-deficient male mice due to enhanced expression of hepatic leptin receptor.

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Yoshino, Satoshi; Satoh, Tetsurou; Yamada, Masanobu; Hashimoto, Koshi; Tomaru, Takuya; Katano-Toki, Akiko; Kakizaki, Satoru; Okada, Shuichi; Shimizu, Hiroyuki; Ozawa, Atsushi; Tuchiya, Takafumi; Ikota, Hayato; Nakazato, Yoichi; Mori, Munemasa; Matozaki, Takashi; Sasaki, Tsutomu; Kitamura, Tadahiro; Mori, Masatomo

2014-09-01

Obesity arises from impaired energy balance, which is centrally coordinated by leptin through activation of the long form of leptin receptor (Leprb). Obesity causes central leptin resistance. However, whether enhanced peripheral leptin sensitivity could overcome central leptin resistance remains obscure. A peripheral metabolic organ targeted by leptin is the liver, with low Leprb expression. We here show that mice fed a high-fat diet (HFD) and obese patients with hepatosteatosis exhibit increased expression of hepatic helicase with zinc finger 2, a transcriptional coactivator (Helz2), which functions as a transcriptional coregulator of several nuclear receptors, including peroxisome proliferator-activated receptor γ in vitro. To explore the physiological importance of Helz2, we generated Helz2-deficient mice and analyzed their metabolic phenotypes. Helz2-deficient mice showing hyperleptinemia associated with central leptin resistance were protected against HFD-induced obesity and had significantly up-regulated hepatic Leprb expression. Helz2 deficiency and adenovirus-mediated liver-specific exogenous Leprb overexpression in wild-type mice significantly stimulated hepatic AMP-activated protein kinase on HFD, whereas Helz2-deficient db/db mice lacking functional Leprb did not. Fatty acid-β oxidation was increased in Helz2-deficeint hepatocytes, and Helz2-deficient mice revealed increased oxygen consumption and decreased respiratory quotient in calorimetry analyses. The enhanced hepatic AMP-activated protein kinase energy-sensing pathway in Helz2-deficient mice ameliorated hyperlipidemia, hepatosteatosis, and insulin resistance by reducing lipogenic gene expression and stimulating lipid-burning gene expression in the liver. These findings together demonstrate that Helz2 deficiency ameliorates HFD-induced metabolic abnormalities by stimulating endogenous hepatic Leprb expression, despite central leptin resistance. Hepatic HELZ2 might be a novel target molecule for

Motif signatures of transcribed enhancers

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Kleftogiannis, Dimitrios

2017-09-14

In mammalian cells, transcribed enhancers (TrEn) play important roles in the initiation of gene expression and maintenance of gene expression levels in spatiotemporal manner. One of the most challenging questions in biology today is how the genomic characteristics of enhancers relate to enhancer activities. This is particularly critical, as several recent studies have linked enhancer sequence motifs to specific functional roles. To date, only a limited number of enhancer sequence characteristics have been investigated, leaving space for exploring the enhancers genomic code in a more systematic way. To address this problem, we developed a novel computational method, TELS, aimed at identifying predictive cell type/tissue specific motif signatures. We used TELS to compile a comprehensive catalog of motif signatures for all known TrEn identified by the FANTOM5 consortium across 112 human primary cells and tissues. Our results confirm that distinct cell type/tissue specific motif signatures characterize TrEn. These signatures allow discriminating successfully a) TrEn from random controls, proxy of non-enhancer activity, and b) cell type/tissue specific TrEn from enhancers expressed and transcribed in different cell types/tissues. TELS codes and datasets are publicly available at http://www.cbrc.kaust.edu.sa/TELS.

Titanium phosphate glass microcarriers induce enhanced osteogenic cell proliferation and human mesenchymal stem cell protein expression

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Nilay J Lakhkar

2015-11-01

Full Text Available In this study, we have developed 50- to 100-µm-sized titanium phosphate glass microcarriers (denoted as Ti5 that show enhanced proliferation of human mesenchymal stem cells and MG63 osteosarcoma cells, as well as enhanced human mesenchymal stem cell expression of bone differentiation markers, in comparison with commercially available glass microspheres at all time points. We also demonstrate that these microcarriers provide superior human mesenchymal stem cell proliferation with conventional Dulbecco’s Modified Eagle medium than with a specially developed commercial stem cell medium. The microcarrier proliferative capacity is revealed by a 24-fold increase in MG63 cell numbers in spinner flask bioreactor studies performed over a 7-day period, versus only a 6-fold increase in control microspheres under the same conditions; the corresponding values of Ti5 and control microspheres under static culture are 8-fold and 7-fold, respectively. The capability of guided osteogenic differentiation is confirmed by ELISAs for bone morphogenetic protein-2 and osteopontin, which reveal significantly greater expression of these markers, especially osteopontin, by human mesenchymal stem cells on the Ti5 microspheres than on the control. Scanning electron microscopy and confocal laser scanning microscopy images reveal favorable MG63 and human mesenchymal stem cell adhesion on the Ti5 microsphere surfaces. Thus, the results demonstrate the suitability of the developed microspheres for use as microcarriers in bone tissue engineering applications.

Enhanced caveolin-1 expression increases migration, anchorage-independent growth and invasion of endometrial adenocarcinoma cells

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Diaz-Valdivia, Natalia; Bravo, Denisse; Huerta, Hernán; Henriquez, Soledad; Gabler, Fernando; Vega, Margarita; Romero, Carmen; Calderon, Claudia; Owen, Gareth I.; Leyton, Lisette; Quest, Andrew F. G.

2015-01-01

Caveolin-1 (CAV1) has been implicated both in tumor suppression and progression, whereby the specific role appears to be context dependent. Endometrial cancer is one of the most common malignancies of the female genital tract; however, little is known about the role of CAV1 in this disease. Here, we first determined by immunohistochemistry CAV1 protein levels in normal proliferative human endometrium and endometrial tumor samples. Then using two endometrial cancer cell lines (ECC: Ishikawa and Hec-1A) we evaluated mRNA and protein levels of CAV1 by real time qPCR and Western blot analysis, respectively. The role of CAV1 expression in ECC malignancy was further studied by either inducing its expression in endometrial cancer cells with the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (4β-TPA) or decreasing expression using short-hairpin RNA constructs, and then evaluating the effects of these changes on ECC proliferation, transmigration, matrigel invasion, and colony formation in soft agar. Immunohistochemical analysis of endometrial epithelia revealed that substantially higher levels of CAV1 were present in endometrial tumors than the normal proliferative epithelium. Also, in Ishikawa and Hec-1A endometrial cancer cells CAV1 expression was readily detectable. Upon treatment with 4β-TPA CAV1 levels increased and coincided with augmented cell transmigration, matrigel invasion, as well as colony formation in soft agar. Reduction of CAV1 expression using short-hairpin RNA constructs ablated these effects in both cell types whether treated or not with 4β-TPA. Alternatively, CAV1 expression appeared not to modulate significantly proliferation of these cells. Our study shows that elevated CAV1, observed in patients with endometrial cancer, is linked to enhanced malignancy of endometrial cancer cells, as evidenced by increased migration, invasion and anchorage-independent growth. The online version of this article (doi:10.1186/s12885-015-1477-5) contains

Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression.

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Hinds, Terry D; Peck, Bailey; Shek, Evan; Stroup, Steven; Hinson, Jennifer; Arthur, Susan; Marino, Joseph S

2016-02-11

Unlike the glucocorticoid receptor α (GRα), GR β (GRβ) has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex) responsiveness. We measured GR isoform expression in C₂C12 muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C₂C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a) mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx) and muscle ring finger 1 (MuRF1) response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids.

Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression

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Terry D. Hinds

2016-02-01

Full Text Available Unlike the glucocorticoid receptor α (GRα, GR β (GRβ has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex responsiveness. We measured GR isoform expression in C2C12 muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C2C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx and muscle ring finger 1 (MuRF1 response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids.

[Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA].

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Jia, Xiao-Wei; Zhang, Guo-Hui; Shi, Hai-Yan

2012-12-01

Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription. We express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction. The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15. kod-ssb may in future be used to enhance DNA and cDNA amplification.

The phosphodiesterase inhibitor, ibudilast, attenuates neuroinflammation in the MPTP model of Parkinson's disease.

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Joanna Schwenkgrub

Full Text Available Since the degeneration of the nigrostriatal dopaminergic pathway in Parkinson's disease (PD is associated with the inflammation process and decreased levels of cyclic nucleotides, inhibition of up-regulated cyclic nucleotide phosphodiesterases (PDEs appears to be a promising therapeutic strategy. We used ibudilast (IBD, a non-selective PDE3,4,10,11 inhibitor, due to the abundant PDE 4 and 10 expression in the striatum. The present study for the first time examined the efficacy of IBD in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP mouse model of PD.IBD [0, 20, 30, 40, or 50 mg/kg] was injected b.i.d. subcutaneously for nine days to three-month-old male C57Bl/10Tar mice, beginning two days prior to MPTP (60 mg/kg intoxication. High-pressure liquid chromatography, Western blot analysis, and real time RT-PCR methods were applied.Our study demonstrated that chronic administration of IBD attenuated astroglial reactivity and increased glial cell-derived neurotrophic factor (GDNF production in the striatum. Moreover, IBD reduced TNF-α, IL-6, and IL-1β expression.IBD had a well-defined effect on astroglial activation in the mouse model of PD; however, there was no protective effect in the acute phase of injury. Diminished inflammation and an increased level of GDNF may provide a better outcome in the later stages of neurodegeneration.

A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

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Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

2016-09-01

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

Adenovirus or HA-2 fusogenic peptide-assisted lipofection increases cytoplasmic levels of plasmid in nondividing endothelium with little enhancement of transgene expression.

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Subramanian, Ajit; Ma, Haiching; Dahl, Kris N; Zhu, Jingya; Diamond, Scott L

2002-01-01

Adenovirus-assisted lipofection has been reported to increase transfection efficiency through mechanisms potentially involving endosome escape and/or nuclear targeting activity. Similarly, transfection with the viral fusogenic peptide HA-2 of the influenza virus hemagglutinin can increase transfection efficiency. However, there are few studies examining the mechanism and intracellular trafficking of these viral and/or viral fusogenic peptide-assisted lipofections. Endosome escape was directly assayed with T7 RNA polymerase bound to plasmid (pTM beta gal) expressing beta-galactosidase under a T7 promoter to detect transcribable plasmid that escapes the endosomal compartment. Lipofection of pTM beta gal with replication-deficient adenovirus (Ad5-null) at a multiplicity of infection (MOI) of 100 and 1000 increased cytoplasmic levels of transcribable plasmid by 24- and 117-fold, respectively, over lipofection alone, without an effect on total plasmid uptake. However, lipofection of pCMV beta gal with Ad5-null at a MOI of 100 and 1000 increased transgene expression only seven- and eight-fold, respectively, over lipofection alone. Thus, a 24-fold increase in endosome escape saturated expression from pCMV beta gal and provided only a seven-fold benefit in nondividing cells, which was not significantly increased with further increases in endosome escape. A cationic form of HA-2 (HA-K(4)) also caused significant enhancements in endosome escape, as detected with the cytoplasmic transcription assay. However, HA-K(4) enhancement of endosome escape did not correlate with transgene expression from pCMV beta gal, consistent with the detection of HA-K(4)-mediated partitioning of plasmid to the insoluble fraction of the cell lysate. These results indicate that enhancement of endosome escape in nondividing cells does not fully alleviate rate limits related to nuclear import of the plasmid. Copyright 2001 John Wiley & Sons, Ltd.

Polymethoxylated flavones potentiate the cytolytic activity of NK leukemia cell line KHYG-1 via enhanced expression of granzyme B.

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Saito, Takeshi; Abe, Daigo; Nogata, Yoichi

2015-01-16

Polymethoxylated flavones (PMFs) are found in the peel tissues of some citrus species. Here, we report that PMFs, such as nobiletin, potentiate the cytolytic activity of KHYG-1 natural killer (NK) leukemia cells. Nobiletin markedly enhanced the expression of granzyme B, a serine protease that plays critical roles in the cytolytic activity of NK cells. The potentiated cytolytic activity induced by nobiletin was canceled by the granzyme B inhibitor Z-AAD-CMK. Nobiletin also increased the levels of phosphorylated CREB, ERK1/2, and p38 MAPK in KHYG-1 cells, which are known to participate in NK cell function. Inhibition of an upstream kinase of ERK1/2 failed to reduce the granzyme B expression and KHYG-1 cytolytic activity. Meanwhile, inhibition of p38 MAPK attenuated both granzyme B expression and KHYG-1 cytolytic activity. These results suggest that the primary role of nobiletin in KHYG-1 cytolytic activity lies in upregulation of granzyme B expression, at least in part, mediated through p38 MAPK function. Copyright © 2014 Elsevier Inc. All rights reserved.

An Ultra-High Fluorescence Enhancement and High Throughput Assay for Revealing Expression and Internalization of Chemokine Receptor CXCR4.

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He, Hua; Wang, Xiaojuan; Cheng, Tiantian; Xia, Yongqing; Lao, Jun; Ge, Baosheng; Ren, Hao; Khan, Naseer Ullah; Huang, Fang

2016-04-18

Revealing chemokine receptor CXCR4 expression, distribution, and internalization levels in different cancers helps to evaluate cancer progression or prognosis and to set personalized treatment strategy. We here describe a sensitive and high-throughput immunoassay for determining CXCR4 expression and distribution in cancer cells. The assay is accessible to a wide range of users in an ordinary lab only by dip-coating poly(styrene-co-N-isopropylacrylamide) spheres on the glass substrate. The self- assembled spheres form three-dimensional photonic colloidal crystals which enhance the fluorescence of CF647 and Alexa Fluor 647 by a factor of up to 1000. CXCR4 in cells is detected by using the sandwich immunoassay, where the primary antibody recognizes CXCR4 and the secondary antibody is labeled with CF647. With the newly established assay, we quantified the total expression of CXCR4, its distribution on the cell membrane and cytoplasm, and revealed their internalization level upon SDF-1α activation in various cancer cells, even for those with extremely low expression level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced regulatory gene expressions in the blood and articular cartilage of patients with rheumatoid arthritis

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Elena Vasilyevna Chetina

2012-01-01

accompanied by the elevated concentrations of the corresponding proteins in the cell lysates of the patients with RA compared to the controls. Conclusion. The findings suggest for the first time that regulatory mTOR, ATG1, p21, and TNFа gene expressions are enhanced in the blood and articular cartilage of RA patients. These changes are accompanied by the increased expression of MMP 13 gene that is responsible for articular cartilage resorption. Therefore, the higher expression of the examined regulatory genes in the blood of RA patients may be indicative of articular cartilage degradation.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

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Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

2009-12-30

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

Science.gov (United States)

2009-01-01

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T

Ectopic phytocystatin expression leads to enhanced drought stress tolerance in soybean (Glycine max) and Arabidopsis thaliana through effects on strigolactone pathways and can also result in improved seed traits.

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Quain, Marian D; Makgopa, Matome E; Márquez-García, Belén; Comadira, Gloria; Fernandez-Garcia, Nieves; Olmos, Enrique; Schnaubelt, Daniel; Kunert, Karl J; Foyer, Christine H

2014-09-01

Ectopic cystatin expression has long been used in plant pest management, but the cysteine protease, targets of these inhibitors, might also have important functions in the control of plant lifespan and stress tolerance that remain poorly characterized. We therefore characterized the effects of expression of the rice cystatin, oryzacystatin-I (OCI), on the growth, development and stress tolerance of crop (soybean) and model (Arabidopsis thaliana) plants. Ectopic OCI expression in soybean enhanced shoot branching and leaf chlorophyll accumulation at later stages of vegetative development and enhanced seed protein contents and decreased the abundance of mRNAs encoding strigolactone synthesis enzymes. The OCI-expressing A. thaliana showed a slow-growth phenotype, with increased leaf numbers and enhanced shoot branching at flowering. The OCI-dependent inhibition of cysteine proteases enhanced drought tolerance in soybean and A. thaliana, photosynthetic CO2 assimilation being much less sensitive to drought-induced inhibition in the OCI-expressing soybean lines. Ectopic OCI expression or treatment with the cysteine protease inhibitor E64 increased lateral root densities in A. thaliana. E64 treatment also increased lateral root densities in the max2-1 mutants that are defective in strigolactone signalling, but not in the max3-9 mutants that are defective in strigolactone synthesis. Taken together, these data provide evidence that OCI-inhibited cysteine proteases participate in the control of growth and stress tolerance through effects on strigolactones. We conclude that cysteine proteases are important targets for manipulation of plant growth, development and stress tolerance, and also seed quality traits. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Transgenic expression of lactoferrin imparts enhanced resistance to head blight of wheat caused by Fusarium graminearum

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Han Jigang

2012-03-01

Full Text Available Abstract Background The development of plant gene transfer systems has allowed for the introgression of alien genes into plant genomes for novel disease control strategies, thus providing a mechanism for broadening the genetic resources available to plant breeders. Using the tools of plant genetic engineering, a broad-spectrum antimicrobial gene was tested for resistance against head blight caused by Fusarium graminearum Schwabe, a devastating disease of wheat (Triticum aestivum L. and barley (Hordeum vulgare L. that reduces both grain yield and quality. Results A construct containing a bovine lactoferrin cDNA was used to transform wheat using an Agrobacterium-mediated DNA transfer system to express this antimicrobial protein in transgenic wheat. Transformants were analyzed by Northern and Western blots to determine lactoferrin gene expression levels and were inoculated with the head blight disease fungus F. graminearum. Transgenic wheat showed a significant reduction of disease incidence caused by F. graminearum compared to control wheat plants. The level of resistance in the highly susceptible wheat cultivar Bobwhite was significantly higher in transgenic plants compared to control Bobwhite and two untransformed commercial wheat cultivars, susceptible Wheaton and tolerant ND 2710. Quantification of the expressed lactoferrin protein by ELISA in transgenic wheat indicated a positive correlation between the lactoferrin gene expression levels and the levels of disease resistance. Conclusions Introgression of the lactoferrin gene into elite commercial wheat, barley and other susceptible cereals may enhance resistance to F. graminearum.

n-Butyl benzyl phthalate promotes breast cancer progression by inducing expression of lymphoid enhancer factor 1.

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Tsung-Hua Hsieh

Full Text Available Environmental hormones play important roles in regulating the expression of genes involved in cell proliferation, drug resistance, and breast cancer risk; however, their precise role in human breast cancer cells during cancer progression remains unclear. To elucidate the effect of the most widely used industrial phthalate, n-butyl benzyl phthalate (BBP, on cancer progression, we evaluated the results of BBP treatment using a whole human genome cDNA microarray and MetaCore software and selected candidate genes whose expression was changed by more than ten-fold by BBP compared with controls to analyze the signaling pathways in human breast cancer initiating cells (R2d. A total of 473 genes were upregulated, and 468 were downregulated. Most of these genes are involved in proliferation, epithelial-mesenchymal transition, and angiogenesis signaling. BBP induced the viability, invasion and migration, and tube formation in vitro, and Matrigel plug angiogenesis in vivo of R2d and MCF-7. Furthermore, the viability and invasion and migration of these cell lines following BBP treatment was reduced by transfection with a small interfering RNA targeting the mRNA for lymphoid enhancer-binding factor 1; notably, the altered expression of this gene consistently differentiated tumors expressing genes involved in proliferation, epithelial-mesenchymal transition, and angiogenesis. These findings contribute to our understanding of the molecular impact of the environmental hormone BBP and suggest possible strategies for preventing and treating human breast cancer.

IFN-γ-producing NKT cells exacerbate sepsis by enhancing C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils.

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Kim, Ji Hyung; Oh, Sae Jin; Ahn, Sehee; Chung, Doo Hyun

2014-07-01

A role for NKT cells has been implicated in sepsis, but the mechanism by which NKT cells contribute to sepsis remains unclear. Here, we examined WT and NKT-cell-deficient mice of C57BL/6 background during cecal ligation and puncture-induced sepsis. The levels of C5a, IFN-γ, and IL-10 were higher in the serum and peritoneal fluid of WT mice than in those of CD1d(-/-) mice, while the mortality rate was lower in CD1d(-/-) mice than in WT mice. C5a blockade decreased mortality of WT mice during sepsis, whereas it did not alter that of CD1d(-/-) mice. As assessed by intracellular staining, NKT cells expressed IFN-γ, while neutrophils expressed IL-10. Upon coculture, IL-10-deficient NKT cells enhanced IL-10 production by WT, but not IFN-γR-deficient, neutrophils. Meanwhile, CD1d(-/-) mice exhibited high CD55 expression on neutrophils during sepsis, whereas those cells from WT mice expressed minimal levels of CD55. Recombinant IL-10 administration into CD1d(-/-) mice reduced CD55 expression on neutrophils. Furthermore, adoptive transfer of sorted WT, but not IFN-γ-deficient, NKT cells into CD1d(-/-) mice suppressed CD55 expression on neutrophils, but increased IL-10 and C5a levels. Taken together, IFN-γ-producing NKT cells enhance C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils, thereby exacerbating sepsis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of the Aeluropus littoralis AlSAP Gene Enhances Rice Yield under Field Drought at the Reproductive Stage

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Thaura Ghneim-Herrera

2017-06-01

Full Text Available We evaluated the yields of Oryza sativa L. ‘Nipponbare’ rice lines expressing a gene encoding an A20/AN1 domain stress-associated protein, AlSAP, from the halophyte grass Aeluropus littoralis under the control of different promoters. Three independent field trials were conducted, with drought imposed at the reproductive stage. In all trials, the two transgenic lines, RN5 and RN6, consistently out-performed non-transgenic (NT and wild-type (WT controls, providing 50–90% increases in grain yield (GY. Enhancement of tillering and panicle fertility contributed to this improved GY under drought. In contrast with physiological records collected during previous greenhouse dry-down experiments, where drought was imposed at the early tillering stage, we did not observe significant differences in photosynthetic parameters, leaf water potential, or accumulation of antioxidants in flag leaves of AlSAP-lines subjected to drought at flowering. However, AlSAP expression alleviated leaf rolling and leaf drying induced by drought, resulting in increased accumulation of green biomass. Therefore, the observed enhanced performance of the AlSAP-lines subjected to drought at the reproductive stage can be tentatively ascribed to a primed status of the transgenic plants, resulting from a higher accumulation of biomass during vegetative growth, allowing reserve remobilization and maintenance of productive tillering and grain filling. Under irrigated conditions, the overall performance of AlSAP-lines was comparable with, or even significantly better than, the NT and WT controls. Thus, AlSAP expression inflicted no penalty on rice yields under optimal growth conditions. Our results support the use of AlSAP transgenics to reduce rice GY losses under drought conditions.

Nanocarrier-mediated foliar zinc fertilization influences expression of metal homeostasis related genes in flag leaves and enhances gluten content in durum wheat.

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Deshpande, Paresh; Dapkekar, Ashwin; Oak, Manoj; Paknikar, Kishore; Rajwade, Jyutika

2018-01-01

Wheat is the staple food for most of the world's population; however, it is a poor source of zinc. Foliar fertilization of zinc via zinc loaded chitosan nanocarriers (Zn-CNP) post-anthesis has proved to be a promising approach for grain zinc enhancement in durum wheat as evidenced in our earlier study. However, the molecular mechanism of uptake of zinc via Zn-CNP remains unclear. Foliar application of Zn-CNP was performed at post anthesis stages in two durum wheat cultivars (MACS 3125 and UC1114, containing the Gpc-B1 gene), and expression levels of several metal-related genes were analyzed during early senescence. Zn-CNP application indeed caused changes in gene expression as revealed by qPCR data on representative genes involved in metal homeostasis, phloem transporters, and leaf senescence. Furthermore, zinc-regulated transporters and iron (Fe)-regulated transporter-like protein (ZIP) family [ZIP1, ZIP7, ZIP15], CA (carbonic anhydrase), and DMAS (2'-deoxymugineic acid synthase) in flag leaves exhibited significant correlation with zinc content in the seeds. The analysis of grain endosperm proteins showed enhancement of gamma gliadins while other gluten subunits decreased. Gene expression within ZIP family members varied with the type of cultivar mostly attributed to the Gpc-B1, concentration of external zinc ions as well as the type of tissue analyzed. Correlation analysis revealed the involvement of the selected genes in zinc enhancement. At the molecular level, uptake of zinc via Zn-CNP nanocarrier was comparable to the uptake of zinc via common zinc fertilizers i.e. ZnSO4.

Nanocarrier-mediated foliar zinc fertilization influences expression of metal homeostasis related genes in flag leaves and enhances gluten content in durum wheat.

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Paresh Deshpande

Full Text Available Wheat is the staple food for most of the world's population; however, it is a poor source of zinc. Foliar fertilization of zinc via zinc loaded chitosan nanocarriers (Zn-CNP post-anthesis has proved to be a promising approach for grain zinc enhancement in durum wheat as evidenced in our earlier study. However, the molecular mechanism of uptake of zinc via Zn-CNP remains unclear.Foliar application of Zn-CNP was performed at post anthesis stages in two durum wheat cultivars (MACS 3125 and UC1114, containing the Gpc-B1 gene, and expression levels of several metal-related genes were analyzed during early senescence. Zn-CNP application indeed caused changes in gene expression as revealed by qPCR data on representative genes involved in metal homeostasis, phloem transporters, and leaf senescence. Furthermore, zinc-regulated transporters and iron (Fe-regulated transporter-like protein (ZIP family [ZIP1, ZIP7, ZIP15], CA (carbonic anhydrase, and DMAS (2'-deoxymugineic acid synthase in flag leaves exhibited significant correlation with zinc content in the seeds. The analysis of grain endosperm proteins showed enhancement of gamma gliadins while other gluten subunits decreased. Gene expression within ZIP family members varied with the type of cultivar mostly attributed to the Gpc-B1, concentration of external zinc ions as well as the type of tissue analyzed. Correlation analysis revealed the involvement of the selected genes in zinc enhancement.At the molecular level, uptake of zinc via Zn-CNP nanocarrier was comparable to the uptake of zinc via common zinc fertilizers i.e. ZnSO4.

Enhancement of antiproliferative activity of interferons by RNA interference-mediated silencing of SOCS gene expression in tumor cells.

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Takahashi, Yuki; Kaneda, Haruka; Takasuka, Nana; Hattori, Kayoko; Nishikawa, Makiya; Watanabe, Yoshihiko; Takakura, Yoshinobu

2008-08-01

The suppressor of cytokine signaling (SOCS) proteins, negative regulators of interferon (IFN)-induced signaling pathways, is involved in IFN resistance of tumor cells. To improve the growth inhibitory effect of IFN-beta and IFN-gamma on a murine melanoma cell line, B16-BL6, and a murine colon carcinoma cell line, Colon26 cells, SOCS-1 and SOCS-3 gene expression in tumor cells was downregulated by transfection of plasmid DNA expressing short hairpin RNA targeting one of these genes (pshSOCS-1 and pshSOCS-3, respectively). Transfection of pshSOCS-1 significantly increased the antiproliferative effect of IFN-gamma on B16-BL6 cells. However, any other combinations of plasmids and IFN had little effect on the growth of B16-BL6 cells. In addition, transfection of pshSOCS-1 and pshSOCS-3 produced little improvement in the effect of IFN on Colon26 cells. To understand the mechanism underlining these findings, the level of SOCS gene expression was measured by real time polymerase chain reaction. Addition of IFN-gamma greatly increased the SOCS-1 mRNA expression in B16-BL6 cells. Taking into account the synergistic effect of pshSOCS-1 and IFN-gamma on the growth of B16-BL6 cells, these findings suggest that IFN-gamma-induced high SOCS-1 gene expression in B16-BL6 cells significantly interferes with the antiproliferative effect of IFN-gamma. These results indicate that silencing SOCS gene expression can be an effective strategy to enhance the antitumor effect of IFN under conditions in which the SOCS gene expression is upregulated by IFN.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

OpenAIRE

Matsumoto, M; Imagawa, M; Aoki, Y

2000-01-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did no...

Increased expression of enhancer of Zeste Homolog 2 (EZH2) differentiates squamous cell carcinoma from normal skin and actinic keratosis.

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Xie, Qiang; Wang, Hongbei; Heilman, Edward R; Walsh, Michael G; Haseeb, M A; Gupta, Raavi

2014-01-01

Enhancer of Zeste Homolog 2 (EZH2) is a polycomb group protein that has been shown to be involved in the progression of multiple human cancers including melanoma. The expression of EZH2 in normal skin and in pre-malignant and malignant cutaneous squamous cell carcinoma (SCC) has not been studied. We examined the expression of EZH2 in normal skin, actinic keratosis (AK), SCC in situ, well-differentiated (SCC-WD), moderately-differentiated (SCC-MD) and poorly-differentiated SCC (SCC-PD) to ascertain whether EZH2 expression differentiates these conditions. Immunohistochemical staining for EZH2 was performed on formalin-fixed paraffin-embedded biopsies and a tissue microarray containing normal skin, AK, SCC in situ, and SCC of different grades. In comparison to the normal skin, EZH2 expression in actinic keratosis was increased (p=0.03). Similarly, EZH2 expression in all of the neoplastic conditions studied (SCC in situ, SCC-WD, SCC-MD and SCC-PD) was greatly increased in comparison to both the normal skin and actinic keratosis (p≤0.001). EZH2 expression increases incrementally from normal skin to AK and further to SCC, suggesting a role for EZH2 in the progression and differentiation of SCC. EZH2 expression may be used as a diagnostic marker for differentiating SCC from AK or normal skin.

Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons

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Fabio eTescarollo

2014-08-01

Full Text Available The excitatory neurotransmitter glutamate has been reported to have a major impact on brain energy metabolism. Using primary cultures of rat hippocampal neurons, we observed that glutamate reduces glucose utilization in this cell type, suggesting alteration in mitochondrial oxidative metabolism. The aquaglyceroporin AQP9 and the monocarboxylate transporter MCT2, two transporters for oxidative energy substrates, appear to be present in mitochondria of these neurons. Moreover, they not only co-localize but they interact with each other as they were found to co-immunoprecipitate from hippocampal neuron homogenates. Exposure of cultured hippocampal neurons to glutamate 100 µM for 1 hour led to enhanced expression of both AQP9 and MCT2 at the protein level without any significant change at the mRNA level. In parallel, a similar increase in the protein expression of LDHA was evidenced without an effect on the mRNA level. These data suggest that glutamate exerts an influence on neuronal energy metabolism likely through a regulation of the expression of some key mitochondrial proteins.

The nigrostriatal dopamine system of aging GFRα-1 heterozygous mice: neurochemistry, morphology and behavior

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Zaman, Vandana; Boger, Heather A.; Granholm, Ann-Charlotte; Rohrer, Baerbel; Moore, Alfred; Buhusi, Mona; Gerhardt, Greg A.; Hoffer, Barry J.; Middaugh, Lawrence D.

2009-01-01

Given the established importance of glial cell line-derived neurotrophic factor (GDNF) in maintaining dopaminergic neurotransmitter systems, the nigrostriatal system and associated behaviors of mice with genetic reduction of its high-affinity receptor, GDNF receptor (GFR)α-1 (GFRα-1+/−), were compared with wild-type controls. Motor activity and the stimulatory effects of a dopamine (DA) D1 receptor agonist (SKF 82958) were assessed longitudinally at 8 and 18 months of age. Monoamine concentrations and dopaminergic nerve terminals in the striatum and the number of dopaminergic neurons in the substantia nigra (SN) were assessed. The results support the importance of GFRα-1 in maintaining normal function of the nigrostriatal dopaminergic system, with deficits being observed for GFRα-1+/− mice at both ages. Motor activity was lower and the stimulatory effects of the DA agonist were enhanced for the older GFRα-1+/− mice. DA in the striatum was reduced in the GFRα-1+/− mice at both ages, and tyrosine hydroxylase-positive cell numbers in the SN were reduced most substantially in the older GFRα-1+/− mice. The combined behavioral, pharmacological probe, neurochemical and morphological measures provide evidence of abnormalities in GFRα-1+/− mice that are indicative of an exacerbated aging-related decline in dopaminergic system function. The noted deficiencies, in turn, suggest that GFRα-1 is necessary for GDNF to maintain normal function of the nigrostriatal dopaminergic system. Although the precise mechanism(s) for the aging-related changes in the dopaminergic system remain to be established, the present study clearly establishes that genetic reductions in GFRα-1 can contribute to the degenerative changes observed in this system during the aging process. PMID:18973577

Enhanced water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene.

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Amara, Imen; Capellades, Montserrat; Ludevid, M Dolors; Pagès, Montserrat; Goday, Adela

2013-06-15

Late Embryogenesis Abundant (LEA) proteins participate in plant stress responses and contribute to the acquisition of desiccation tolerance. In this report Rab28 LEA gene has been over-expressed in maize plants under a constitutive maize promoter. The expression of Rab28 transcripts led to the accumulation and stability of Rab28 protein in the transgenic plants. Native Rab28 protein is localized to nucleoli in wild type maize embryo cells; here we find by whole-mount immunocytochemistry that in root cells of Rab28 transgenic and wild-type plants the protein is also associated to nucleolar structures. Transgenic plants were tested for stress tolerance and resulted in sustained growth under polyethyleneglycol (PEG)-mediated dehydration compared to wild-type controls. Under osmotic stress transgenic seedlings showed increased leaf and root areas, higher relative water content (RWC), reduced chlorophyll loss and lower Malondialdehyde (MDA) production in relation to wild-type plants. Moreover, transgenic seeds exhibited higher germination rates than wild-type seeds under water deficit. Overall, our results highlight the presence of transgenic Rab28 protein in nucleolar structures and point to the potential of group 5 LEA Rab28 gene as candidate to enhance stress tolerance in maize plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

Preventive effects of a fermented dairy product against Alzheimer's disease and identification of a novel oleamide with enhanced microglial phagocytosis and anti-inflammatory activity.

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Yasuhisa Ano

Full Text Available Despite the ever-increasing number of patients with dementia worldwide, fundamental therapeutic approaches to this condition have not been established. Epidemiological studies suggest that intake of fermented dairy products prevents cognitive decline in the elderly. However, the active compounds responsible for the effect remain to be elucidated. The present study aims to elucidate the preventive effects of dairy products on Alzheimer's disease and to identify the responsible component. Here, in a mouse model of Alzheimer's disease (5xFAD, intake of a dairy product fermented with Penicillium candidum had preventive effects on the disease by reducing the accumulation of amyloid β (Aβ and hippocampal inflammation (TNF-α and MIP-1α production, and enhancing hippocampal neurotrophic factors (BDNF and GDNF. A search for preventive substances in the fermented dairy product identified oleamide as a novel dual-active component that enhanced microglial Aβ phagocytosis and anti-inflammatory activity towards LPS stimulation in vitro and in vivo. During the fermentation, oleamide was synthesized from oleic acid, which is an abundant component of general dairy products owing to lipase enzymatic amidation. The present study has demonstrated the preventive effect of dairy products on Alzheimer's disease, which was previously reported only epidemiologically. Moreover, oleamide has been identified as an active component of dairy products that is considered to reduce Aβ accumulation via enhanced microglial phagocytosis, and to suppress microglial inflammation after Aβ deposition. Because fermented dairy products such as camembert cheese are easy to ingest safely as a daily meal, their consumption might represent a preventive strategy for dementia.

Preventive effects of a fermented dairy product against Alzheimer's disease and identification of a novel oleamide with enhanced microglial phagocytosis and anti-inflammatory activity.

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Ano, Yasuhisa; Ozawa, Makiko; Kutsukake, Toshiko; Sugiyama, Shinya; Uchida, Kazuyuki; Yoshida, Aruto; Nakayama, Hiroyuki

2015-01-01

Despite the ever-increasing number of patients with dementia worldwide, fundamental therapeutic approaches to this condition have not been established. Epidemiological studies suggest that intake of fermented dairy products prevents cognitive decline in the elderly. However, the active compounds responsible for the effect remain to be elucidated. The present study aims to elucidate the preventive effects of dairy products on Alzheimer's disease and to identify the responsible component. Here, in a mouse model of Alzheimer's disease (5xFAD), intake of a dairy product fermented with Penicillium candidum had preventive effects on the disease by reducing the accumulation of amyloid β (Aβ) and hippocampal inflammation (TNF-α and MIP-1α production), and enhancing hippocampal neurotrophic factors (BDNF and GDNF). A search for preventive substances in the fermented dairy product identified oleamide as a novel dual-active component that enhanced microglial Aβ phagocytosis and anti-inflammatory activity towards LPS stimulation in vitro and in vivo. During the fermentation, oleamide was synthesized from oleic acid, which is an abundant component of general dairy products owing to lipase enzymatic amidation. The present study has demonstrated the preventive effect of dairy products on Alzheimer's disease, which was previously reported only epidemiologically. Moreover, oleamide has been identified as an active component of dairy products that is considered to reduce Aβ accumulation via enhanced microglial phagocytosis, and to suppress microglial inflammation after Aβ deposition. Because fermented dairy products such as camembert cheese are easy to ingest safely as a daily meal, their consumption might represent a preventive strategy for dementia.

Methamphetamine abuse affects gene expression in brain-derived microglia of SIV-infected macaques to enhance inflammation and promote virus targets

KAUST Repository

Najera, Julia A.

2016-04-23

Background Methamphetamine (Meth) abuse is a major health problem linked to the aggravation of HIV- associated complications, especially within the Central Nervous System (CNS). Within the CNS, Meth has the ability to modify the activity/function of innate immune cells and increase brain viral loads. Here, we examined changes in the gene expression profile of neuron-free microglial cell preparations isolated from the brain of macaques infected with the Simian Immunodeficiency Virus (SIV), a model of neuroAIDS, and exposed to Meth. We aimed to identify molecular patterns triggered by Meth that could explain the detection of higher brain viral loads and the development of a pro-inflammatory CNS environment in the brain of infected drug abusers. Results We found that Meth alone has a strong effect on the transcription of genes associated with immune pathways, particularly inflammation and chemotaxis. Systems analysis led to a strong correlation between Meth exposure and enhancement of molecules associated with chemokines and chemokine receptors, especially CXCR4 and CCR5, which function as co-receptors for viral entry. The increase in CCR5 expression was confirmed in the brain in correlation with increased brain viral load. Conclusions Meth enhances the availability of CCR5-expressing cells for SIV in the brain, in correlation with increased viral load. This suggests that Meth is an important factor in the susceptibility to the infection and to the aggravated CNS inflammatory pathology associated with SIV in macaques and HIV in humans.

Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight.

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Karmakar, Subhasis; Molla, Kutubuddin Ali; Chanda, Palas K; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

2016-01-01

Green tissue-specific simultaneous overexpression of two defense-related genes ( OsCHI11 & OsOXO4 ) in rice leads to significant resistance against sheath blight pathogen ( R. solani ) without distressing any agronomically important traits. Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, P D54O-544 . Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice.

Increased Expression of the Na,K-ATPase alpha4 Isoform Enhances Sperm Motility in Transgenic Mice1

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Jimenez, Tamara; Sanchez, Gladis; McDermott, Jeffrey P.; Nguyen, Anh-Nguyet; Kumar, T. Rajendra; Blanco, Gustavo

2010-01-01

The Na,K-ATPase alpha4 (ATP1A4) isoform is specifically expressed in male germ cells and is highly prevalent in spermatozoa. Although selective inhibition of alpha4 activity with ouabain has been shown to affect sperm motility, a more direct analysis of the role of this isoform in sperm movement has not yet been demonstrated. To establish this, we engineered transgenic mice that express the rat alpha4 isoform fused to green fluorescent protein in male germ cells, under the control of the mouse protamine 1 promoter. We showed that the rat Atp1a4 transgene is expressed in mouse spermatozoa and that it is localized to the sperm flagellum. In agreement with increased expression of the alpha4 isoform, sperm from transgenic mice displayed higher alpha4-specific Na,K-ATPase activity and binding of fluorescently labeled ouabain than wild-type mice. In contrast, expression and activity of ATP1A1 (alpha1), the other Na,K-ATPase alpha isoform present in sperm, remained unchanged. Similar to wild-type mice, mice expressing the alpha4 transgene exhibited normal testis and sperm morphology and no differences in fertility. However, compared to wild-type mice, sperm from transgenic mice displayed plasma membrane hyperpolarization and higher total and progressive motility. Other parameters of motility also increased, including straight-line, curvilinear, and average path velocities and amplitude of lateral head displacement. In addition, sperm from the transgenic mice showed enhanced sperm hyperactive motility, but no changes in progesterone-induced acrosome reaction. Altogether, these results provide new genetic evidence for the role of the ATP1A4 isoform in sperm motility, under both noncapacitating and capacitating conditions. PMID:20826726

Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

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Borges, L G; Seifert, R A; Grant, F J; Hart, C E; Disteche, C M; Edelhoff, S; Solca, F F; Lieberman, M A; Lindner, V; Fischer, E H; Lok, S; Bowen-Pope, D F

1996-09-01

We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

[Small interfering RNA-mediated COX-2 gene silencing enhances chemosensitivity of KB/VCR cells by suppressing MDR-1 gene expression and P-glycoprotein activity].

Science.gov (United States)

Mo, Xianchao; Li, Weizhong

2014-05-01

To investigate the effect of small interfering RNA (siRNA)-mediated COX-2 gene silencing in enhancing the chemosensitivity of KB/VCR cell lines. KB/VCR cells were trasnfected with COX-2 siRNA were examined for expressions of COX-2 and MDR-1 mRNAs with RT-PCR and for Rho-123 accumulation using flow cytometry. MTT assay was used to analyze the proliferation of the transfected KB/VCR cells. Compared with the negative and blank control groups, COX-2 siRNA transfection resulted in significant growth inhibition of KB/VCR cells exposed to vincristine (PKB/VCR cells. COX-2 gene silencing can enhance the chemosensitivity of KB/VCR cells to vincristine, the mechanism of which may involve down-regulated MDR-1 gene expression and inhibition of P-glycoprotein activity.

Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression

International Nuclear Information System (INIS)

Chang, C.-J.; Hsu, C.-C.; Yung, M.-C.; Chen, K.-Y.; Tzao Ching; Wu, W.-F.; Chou, H.-Y.; Lee, Y.-Y.; Lu, K.-H.; Chiou, S.-H.; Ma, H.-I

2009-01-01

CD133-expressing glioma cells play a critical role in tumor recovery after treatment and are resistant to radiotherapy. Herein, we demonstrated that glioblastoma-derived CD133-positive cells (GBM-CD133 + ) are capable of self-renewal and express high levels of embryonic stem cell genes and SirT1 compared to GBM-CD133 - cells. To evaluate the role of SirT1 in GBM-CD133 + , we used a lentiviral vector expressing shRNA to knock-down SirT1 expression (sh-SirT1) in GBM-CD133 + . Silencing of SirT1 significantly enhanced the sensitivity of GBM-CD133 + to radiation and increased the level of radiation-mediated apoptosis. Importantly, knock-down of SirT1 increased the effectiveness of radiotherapy in the inhibition of tumor growth in nude mice transplanted with GBM-CD133 + . Kaplan-Meier survival analysis indicated that the mean survival rate of GBM-CD133 + mice treated with radiotherapy was significantly improved by Sh-SirT1 as well. In sum, these results suggest that SirT1 is a potential target for increasing the sensitivity of GBM and glioblastoma-associated cancer stem cells to radiotherapy.

Functional enhancement of AT1R potency in the presence of the TPαR is revealed by a comprehensive 7TM receptor co-expression screen.

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Jonas Tind Hansen

Full Text Available BACKGROUND: Functional cross-talk between seven transmembrane (7TM receptors can dramatically alter their pharmacological properties, both in vitro and in vivo. This represents an opportunity for the development of novel therapeutics that potentially target more specific biological effects while causing fewer adverse events. Although several studies convincingly have established the existence of 7TM receptor cross-talk, little is known about the frequencey and biological significance of this phenomenon. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the extent of synergism in 7TM receptor signaling, we took a comprehensive approach and co-expressed 123 different 7TM receptors together with the angiotensin II type 1 receptor (AT1R and analyzed how each receptor affected the angiotensin II (AngII response. To monitor the effect we used integrative receptor activation/signaling assay called Receptor Selection and Amplification Technology (R-SAT. In this screen the thromboxane A2α receptor (TPαR was the only receptor which significantly enhanced the AngII-mediated response. The TPαR-mediated enhancement of AngII signaling was significantly reduced when a signaling deficient receptor mutant (TPαR R130V was co-expressed instead of the wild-type TPαR, and was completely blocked both by TPαR antagonists and COX inhibitors inhibiting formation of thromboxane A2 (TXA2. CONCLUSIONS/SIGNIFICANCE: We found a functional enhancement of AT1R only when co-expressed with TPαR, but not with 122 other 7TM receptors. In addition, the TPαR must be functionally active, indicating the AT1R enhancement is mediated by a paracrine mechanism. Since we only found one receptor enhancing AT1R potency, our results suggest that functional augmentation through 7TM receptor cross-talk is a rare event that may require specific conditions to occur.

Viral Vector Mediated Over-Expression of Estrogen Receptor–α in Striatum Enhances the Estradiol-induced Motor Activity in Female Rats and Estradiol Modulated GABA Release

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Schultz, Kristin N.; von Esenwein, Silke A.; Hu, Ming; Bennett, Amy L.; Kennedy, Robert T.; Musatov, Sergei; Toran-Allerand, C. Dominique; Kaplitt, Michael G.; Young, Larry J.; Becker, Jill B.

2009-01-01

Classical estrogen receptor signaling mechanisms involve estradiol binding to intracellular nuclear receptors (estrogen receptor-α (ERα) and estrogen receptor-β (ERβ)) to promote changes in protein expression. Estradiol can also exert effects within seconds to minutes, however, a timescale incongruent with genomic signaling. In the brain, estradiol rapidly potentiates stimulated dopamine release in the striatum of female rats and enhances spontaneous rotational behavior. Furthermore, estradiol rapidly attenuates the K+- evoked increase of GABA in dialysate. We hypothesize that these rapid effects of estradiol in the striatum are mediated by ERα located on the membrane of medium spiny GABAergic neurons. This experiment examined whether over-expression of ERα in the striatum would enhance the effect of estradiol on rotational behavior and the K+- evoked increase in GABA in dialysate. Ovariectomized female rats were tested for rotational behavior or underwent microdialysis experiments after unilateral intrastriatal injections of a recombinant adeno-associated virus (AAV) containing the human ERα cDNA (AAV.ERα) into the striatum; controls received either the same vector into areas outside the striatum or an AAV containing the human alkaline phosphatase gene into the striatum (AAV.ALP). Animals that received AAV.ERα in the striatum exhibited significantly greater estradiol-induced contralateral rotations compared to controls and exhibited behavioral sensitization of contralateral rotations induced by a low dose of amphetamine. ERα over-expression also enhanced the inhibitory effect of estradiol on K+- evoked GABA release suggesting that disinhibition of dopamine release from terminals in the striatum resulted in the enhanced rotational behavior. PMID:19211896

Tamm-Horsfall Glycoprotein Enhances PMN Phagocytosis by Binding to Cell Surface-Expressed Lactoferrin and Cathepsin G That Activates MAP Kinase Pathway

Directory of Open Access Journals (Sweden)

Chia-Li Yu

2011-03-01

Full Text Available The molecular basis of polymorphonuclear neutrophil (PMN phagocytosis-enhancing activity (PEA by human purified urinary Tamm-Horsfall glyco- protein (THP has not been elucidated. In this study, we found human THP bound to lactoferrin (LF and cathepsin G (CG expressed on the surface of PMN, identified by a proteomic study with MALDI-TOF- LC/LC/mass spectrometric analysis. Pre-incubation of 10% SDS-PAGE electrophoresed PMN lysates with monoclonal anti-LF or anti-CG antibody reduced the binding with THP. To elucidate the signaling pathway of THP on PMN activation, we found THP enhanced ERK1/2 phosphorylation, reduced p38 MAP kinase phosphorylation, but had no effect on DNA binding of the five NF-kB family members in PMN. To further clarify whether the carbohydrate-side chains or protein-core structure in THP molecule is responsible for THP-PEA, THP was cleaved by different degrading enzymes with carbohydrate specificity (neuraminidase and β-galactosidase, protein specificity (V8 protease and proteinase K or glycoconjugate specificity (carboxylpeptidase Y and O-sialoglycoprotein endopeptidase. We clearly demonstrated that the intact protein-core structure in THP molecule was more important for THP-PEA than carbohydrate-side chains. Putting these results together, we conclude that THP adheres to surface-expressed LF and CG on PMN and transduces signaling via the MAP kinase pathway to enhance PMN phagocytosis.

Enhanced expression of Gqα and PLC-β1 proteins contributes to vascular smooth muscle cell hypertrophy in SHR: role of endogenous angiotensin II and endothelin-1.

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Atef, Mohammed Emehdi; Anand-Srivastava, Madhu B

2014-07-01

Vascular Gqα signaling has been shown to contribute to cardiac hypertrophy. In addition, angiotensin II (ANG II) was shown to induce vascular smooth muscle cell (VSMC) hypertrophy through Gqα signaling; however, the studies on the role of Gqα and PLC-β1 proteins in VSMC hypertrophy in animal model are lacking. The present study was therefore undertaken to examine the role of Gqα/PLC-β1 proteins and the signaling pathways in VSMC hypertrophy using spontaneously hypertensive rats (SHR). VSMC from 16-wk-old SHR and not from 12-wk-old SHR exhibited enhanced levels of Gqα/PLC-β1 proteins compared with age-matched Wistar-Kyoto (WKY) rats as determined by Western blotting. However, protein synthesis as determined by [(3)H]leucine incorporation was significantly enhanced in VSMC from both 12- and 16-wk-old SHR compared with VSMC from age-matched WKY rats. Furthermore, the knockdown of Gqα/PLC-β1 in VSMC from 16-wk-old SHR by antisense and small interfering RNA resulted in attenuation of protein synthesis. In addition, the enhanced expression of Gqα/PLC-β1 proteins, enhanced phosphorylation of ERK1/2, and enhanced protein synthesis in VSMC from SHR were attenuated by the ANG II AT1 and endothelin-1 (ET-1) ETA receptor antagonists losartan and BQ123, respectively, but not by the ETB receptor antagonist BQ788. In addition, PD98059 decreased the enhanced expression of Gqα/PLC-β1 and protein synthesis in VSMC from SHR. These results suggest that the enhanced levels of endogenous ANG II and ET-1 through the activation of AT1 and ETA receptors, respectively, and MAP kinase signaling, enhanced the expression of Gqα/PLC-β1 proteins in VSMC from 16-wk-old SHR and result in VSMC hypertrophy. Copyright © 2014 the American Physiological Society.

A new approach to enhance the performance of decision tree for classifying gene expression data.

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Hassan, Md; Kotagiri, Ramamohanarao

2013-12-20

Gene expression data classification is a challenging task due to the large dimensionality and very small number of samples. Decision tree is one of the popular machine learning approaches to address such classification problems. However, the existing decision tree algorithms use a single gene feature at each node to split the data into its child nodes and hence might suffer from poor performance specially when classifying gene expression dataset. By using a new decision tree algorithm where, each node of the tree consists of more than one gene, we enhance the classification performance of traditional decision tree classifiers. Our method selects suitable genes that are combined using a linear function to form a derived composite feature. To determine the structure of the tree we use the area under the Receiver Operating Characteristics curve (AUC). Experimental analysis demonstrates higher classification accuracy using the new decision tree compared to the other existing decision trees in literature. We experimentally compare the effect of our scheme against other well known decision tree techniques. Experiments show that our algorithm can substantially boost the classification performance of the decision tree.

CD86 and beta2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells.

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Podojil, Joseph R; Kin, Nicholas W; Sanders, Virginia M

2004-05-28

Stimulation of CD86 (formerly known as B7-2) and/or the beta2-adrenergic receptor on a CD40 ligand/interleukin-4-activated B cell increased the rate of mature IgG1 transcription. To identify the mechanism responsible for this effect, we determined whether CD86 and/or beta2-adrenergic receptor stimulation regulated transcription factor expression and binding to the 3'-IgH enhancer in vitro and in vivo. We showed that CD86 stimulation increased the nuclear localization of NF-kappaB1 (p50) and phosphorylated RelA (p65) and increased Oct-2 expression and binding to the 3'-IgH enhancer, in a protein kinase C-dependent manner. These effects were lost when CD86-deficient or NF-kappaB1-deficient B cells were used. CD86 stimulation also increased the level of IkappaB-alpha phosphorylation but in a protein kinase C-independent manner. Beta2-adrenergic receptor stimulation increased CREB phosphorylation, OCA-B expression, and OCA-B binding to the 3'-IgH enhancer in a protein kinase A-dependent manner, an effect lost when beta2-adrenergic receptor-deficient B cells were used. Also, the beta2-adrenergic receptor-induced increase in the level of mature IgG1 transcript was lost when OCA-B-deficient B cells were used. These data are the first to show that CD86 stimulation up-regulates the expression of the transcription factor Oct-2 in a protein kinase C- and NF-kappaB1-dependent manner, and that beta2-adrenergic receptor stimulation up-regulates the expression of the coactivator OCA-B in a protein kinase A-dependent manner to cooperate with Oct-2 binding to the 3'-IgH enhancer.

Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel

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The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1– expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and p...

Preventive Effects of a Fermented Dairy Product against Alzheimer’s Disease and Identification of a Novel Oleamide with Enhanced Microglial Phagocytosis and Anti-Inflammatory Activity

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Ano, Yasuhisa; Ozawa, Makiko; Kutsukake, Toshiko; Sugiyama, Shinya; Uchida, Kazuyuki; Yoshida, Aruto; Nakayama, Hiroyuki

2015-01-01

Despite the ever-increasing number of patients with dementia worldwide, fundamental therapeutic approaches to this condition have not been established. Epidemiological studies suggest that intake of fermented dairy products prevents cognitive decline in the elderly. However, the active compounds responsible for the effect remain to be elucidated. The present study aims to elucidate the preventive effects of dairy products on Alzheimer’s disease and to identify the responsible component. Here, in a mouse model of Alzheimer’s disease (5xFAD), intake of a dairy product fermented with Penicillium candidum had preventive effects on the disease by reducing the accumulation of amyloid β (Aβ) and hippocampal inflammation (TNF-α and MIP-1α production), and enhancing hippocampal neurotrophic factors (BDNF and GDNF). A search for preventive substances in the fermented dairy product identified oleamide as a novel dual-active component that enhanced microglial Aβ phagocytosis and anti-inflammatory activity towards LPS stimulation in vitro and in vivo. During the fermentation, oleamide was synthesized from oleic acid, which is an abundant component of general dairy products owing to lipase enzymatic amidation. The present study has demonstrated the preventive effect of dairy products on Alzheimer’s disease, which was previously reported only epidemiologically. Moreover, oleamide has been identified as an active component of dairy products that is considered to reduce Aβ accumulation via enhanced microglial phagocytosis, and to suppress microglial inflammation after Aβ deposition. Because fermented dairy products such as camembert cheese are easy to ingest safely as a daily meal, their consumption might represent a preventive strategy for dementia. PMID:25760987

Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

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Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

2016-01-01

Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

CD70 reverse signaling enhances NK cell function and immunosurveillance in CD27-expressing B-cell malignancies.

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Al Sayed, Mohamad F; Ruckstuhl, Carla A; Hilmenyuk, Tamara; Claus, Christina; Bourquin, Jean-Pierre; Bornhauser, Beat C; Radpour, Ramin; Riether, Carsten; Ochsenbein, Adrian F

2017-07-20

The interaction of the tumor necrosis factor receptor (TNFR) CD27 with its ligand CD70 is an emerging target to treat cancer. CD27 signaling provides costimulatory signals to cytotoxic T cells but also increases the frequency of regulatory T cells. Similar to other TNFR ligands, CD70 has been shown to initiate intracellular signaling pathways (CD70 reverse signaling). CD27 is expressed on a majority of B-cell non-Hodgkin lymphoma, but its role in the immune control of lymphoma and leukemia is unknown. We therefore generated a cytoplasmic deletion mutant of CD27 (CD27-trunc) to study the role of CD70 reverse signaling in the immunosurveillance of B-cell malignancies in vivo. Expression of CD27-trunc on malignant cells increased the number of tumor-infiltrating interferon γ-producing natural killer (NK) cells. In contrast, the antitumoral T-cell response remained largely unchanged. CD70 reverse signaling in NK cells was mediated via the AKT signaling pathway and increased NK cell survival and effector function. The improved immune control by activated NK cells prolonged survival of CD27-trunc-expressing lymphoma-bearing mice. Finally, CD70 reverse signaling enhanced survival and effector function of human NK cells in a B-cell acute lymphoblastic leukemia xenotransplants model. Therefore, CD70 reverse signaling in NK cells contributes to the immune control of CD27-expressing B-cell lymphoma and leukemia. © 2017 by The American Society of Hematology.

TALE-mediated modulation of transcriptional enhancers in vivo.

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Crocker, Justin; Stern, David L

2013-08-01

We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

The +37 kb Cebpa Enhancer Is Critical for Cebpa Myeloid Gene Expression and Contains Functional Sites that Bind SCL, GATA2, C/EBPα, PU.1, and Additional Ets Factors

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Cooper, Stacy; Guo, Hong; Friedman, Alan D.

2015-01-01

The murine Cebpa gene contains an evolutionarily conserved 453 bp enhancer located at +37 kb that, together with its promoter, directs expression to myeloid progenitors and to long-term hematopoietic stem cells in transgenic mice. In human acute myeloid leukemia cases, the enhancer lacks point mutations but binds the RUNX1-ETO oncoprotein. The enhancer contains the H3K4me1 and H3K27Ac histone modifications, denoting an active enhancer, at progressively increasing levels as long-term hematopoietic stem cells transition to granulocyte-monocyte progenitors. We previously identified four enhancer sites that bind RUNX1 and demonstrated that their integrity is required for maximal enhancer activity in 32Dcl3 myeloid cells. The +37 kb Cebpa enhancer also contains C/EBP, Ets factor, Myb, GATA, and E-box consensus sites conserved in the human +42 kb CEBPA enhancer. Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells. In 293T gel shift assays, exogenous C/EBPα binds both C/EBP sites, c-Myb binds the Myb site, PU.1 binds the second Ets site, PU.1, Fli-1, ERG, and Ets1 bind the sixth Ets site, GATA2 binds both GATA sites, and SCL binds the second E-box. Endogenous hematopoietic RUNX1, PU.1, Fli-1, ERG, C/EBPα, GATA2, and SCL were previously shown to bind the enhancer, and we find that endogenous PU.1 binds the second Ets site in 32Dcl3 cells. Using CRISPR/Cas9, we developed 32Dcl3 lines in which the wild-type enhancer alleles are replaced with a variant mutant in the seven Ets sites. These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression. In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid transformation. PMID:25938608

Down-regulation of the expression of CCAAT/enhancer binding protein α gene in cervical squamous cell carcinoma

International Nuclear Information System (INIS)

Pan, Zemin; Shao, Renfu; Zheng, Weinan; Zhang, Jinli; Gao, Rui; Li, Dongmei; Guo, Xiaoqing; Han, Hu; Li, Feng; Qu, Shen

2014-01-01

Cervical carcinoma is the second most common cancer and is an important cause of death in women worldwide. CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors that regulate cellular differentiation and proliferation in a variety of tissues. However, the role of C/EBPα gene in cervical cancer is still not clear. We investigated the expression of C/EBPα gene in cervical squamous cell carcinoma. C/EBPα mRNA level was measured by real-time quantitative RT-PCR in cervical cancer tissues and their adjacent normal tissues. C/EBPα protein level was measured by immunohistochemistry. Methylation in the promoter of C/EBPα gene was detected by MALDI TOF MassARRAY. We transfected HeLa cells with C/EBPα expression vector. C/EBPα expression in HeLa cells was examined and HeLa cell proliferation was measured by MTT assay and HeLa cells migration was analyzed by matrigel-coated transwell migration assays. There were significant difference in C/EBPα protein expression between chronic cervicitis and cervical carcinoma (P < 0.001). CEBPα mRNA level was significantly lower in cervical cancer tissues than in normal cervical tissues (P < 0.01). Methylation of the promoter of CEBPα gene in CpG 5, CpG-14.15, CpG-19.20 were significantly higher in cervical cancer than in normal cervical tissues (P < 0.05, P < 0.01, P < 0.05, respectively). CEBPα pcDNA3.1 construct transfected into HeLa cells inhibited cell proliferation and decreased cell migration. Our results indicate that reduced C/EBPα gene expression may play a role in the development of cervical squamous cell carcinoma

Nonverbal and verbal emotional expression and health.

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Berry, D S; Pennebaker, J W

1993-01-01

The spontaneous nonverbal expression of emotion is related to immediate reductions in autonomic nervous system activity. Similar changes in specific autonomic channels occur when individuals are encouraged to verbally express their emotions. Indeed, these physiological changes are most likely to occur among individuals who are either verbally or nonverbally highly expressive. These data suggest that when individuals must actively inhibit emotional expression, they are at increased risk for a variety of health problems. Several experiments are summarized which indicate that verbally expressing traumatic experiences by writing or talking improves physical health, enhances immune function, and is associated with fewer medical visits. Although less research is available regarding nonverbal expression, it is also likely that the nonverbal expression of emotion bears some relation to health status. We propose that the effectiveness of many common expressive therapies (e.g., art, music, cathartic) would be enhanced if clients are encouraged to both express their feelings nonverbally and to put their experiences into words.

Effect of glial cell line-derived neurotrophic factor on retinal function after experimental branch retinal vein occlusion

DEFF Research Database (Denmark)

Ejstrup, Rasmus; Dornonville de la Cour, Morten; Kyhn, Maria Voss

2012-01-01

The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs.......The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs....

Nutrients can enhance the abundance and expression of alkane hydroxylase CYP153 gene in the rhizosphere of ryegrass planted in hydrocarbon-polluted soil.

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Muhammad Arslan

Full Text Available Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination.

Nutrients Can Enhance the Abundance and Expression of Alkane Hydroxylase CYP153 Gene in the Rhizosphere of Ryegrass Planted in Hydrocarbon-Polluted Soil

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Arslan, Muhammad; Afzal, Muhammad; Amin, Imran; Iqbal, Samina; Khan, Qaiser M.

2014-01-01

Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K) in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum) was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination. PMID:25360680

miR-200c targets nuclear factor IA to suppress HBV replication and gene expression via repressing HBV Enhancer I activity.

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Tian, Hui; He, Zhenkun

2018-03-01

Hepatitis B virus (HBV) chronic infection is a health problem in the worldwide, with a underlying higher risk of liver cirrhosis and hepaticocellular carcinoma. A number of studies indicate that microRNAs (miRNAs) play vital roles in HBV replication. This study was designed to explore the potential molecular mechanism of miR-200c in HBV replication. The expression of miR-200c, nuclear factor IA (NFIA) mRNA, HBV DNA, and HBV RNA (pregenomic RNA (pgRNA), and total RNA) were measured by qRCR. The levels of HBsAg and HBeAg were detected by ELISA. NFIA expression at protein level was measured by western blot. The direct interaction between miR-200c and NFIA were identified by Targetscan software and Dual-Luciferase reporter analysis. Enhance I activity were detected by Dual-Luciferase reporter assay. miR-200c expression was prominently reduced in pHBV1.3-tranfected Huh7 and in stable HBV-producing cell line (HepG2.2.15). The enforced expression of miR-200c significantly suppressed HBV replication, as demonstrated by the reduced levels of HBV protein (HBsAg and HBeAg) and, DNA and RNA (pgRNA and total RNA) levels. NFIA was proved to be a target of miR-200c and NFIA overexpression notably stimulated HBV replication. In addition, the inhibitory effect of miR-200c on HBV Enhance I activity was abolished following restoration of NFIA. miR-200c repressed HBV replication by directly targeting NFIA, which might provide a novel therapeutic target for HBV infection. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Coffee enhances the expression of chaperones and antioxidant proteins in rats with nonalcoholic fatty liver disease.

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Salomone, Federico; Li Volti, Giovanni; Vitaglione, Paola; Morisco, Filomena; Fogliano, Vincenzo; Zappalà, Agata; Palmigiano, Angelo; Garozzo, Domenico; Caporaso, Nicola; D'Argenio, Giuseppe; Galvano, Fabio

2014-06-01

Coffee consumption is inversely related to the degree of liver injury in patients with nonalcoholic fatty liver disease (NAFLD). Molecular mediators contributing to coffee's beneficial effects in NAFLD remain to be elucidated. In this study, we administrated decaffeinated espresso coffee or vehicle to rats fed an high-fat diet (HFD) for 12 weeks and examined the effects of coffee on liver injury by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) proteomic analysis combined with mass spectrometry. Rats fed an HFD and water developed panacinar steatosis, lobular inflammation, and mild fibrosis, whereas rats fed an HFD and coffee exhibited only mild steatosis. Coffee consumption increased liver expression of the endoplasmic reticulum chaperones glucose-related protein 78 and protein disulfide-isomerase A3; similarly, coffee drinking enhanced the expression of the mitochondrial chaperones heat stress protein 70 and DJ-1. Furthermore, in agreement with reduced hepatic levels of 8-isoprostanes and 8-hydroxy-2'-deoxyguanosine, proteomic analysis showed that coffee consumption induces the expression of master regulators of redox status (i.e., peroxiredoxin 1, glutathione S-transferase α2, and D-dopachrome tautomerase). Last, proteomics revealed an association of coffee intake with decreased expression of electron transfer flavoprotein subunit α, a component of the mitochondrial respiratory chain, involved in de novo lipogenesis. In this study, we were able to identify by proteomic analysis the stress proteins mediating the antioxidant effects of coffee; moreover, we establish for the first time the contribution of specific coffee-induced endoplasmic reticulum and mitochondrial chaperones ensuring correct protein folding and degradation in the liver. Copyright © 2014 Mosby, Inc. All rights reserved.

Over-expression of CXCR4, a stemness enhancer, in human blastocysts by low level laser irradiation

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Mohammad Hossein Tahmasbi

2013-09-01

Full Text Available The key role of chemokine receptor CXCR4 in the maintenance of stemness property of stem cells has been shown recently. The low level laser irradiation (LLLI is being used currently in a wide variety of clinical cases as a therapeutic tool for wound healing, relieving pain and destroying tumor cells. The aim of this study was to evaluate the effect of LLLI mimicking low level laser therapy (LLLT on the expression level of CXCR4 gene a few hours after irradiation on human blastocysts. After the development of human embryos to the first grade blastocyst stage, they were irradiated with a low power Ga-Al-As laser at a continuous wavelength of 650 nm and a power output of 30 mW. The total RNA of the irradiated blastocysts and control groups were isolated in groups of 1x2 J/cm2, 2x2 J/cm2, 1x4 J/cm2 and 2x4 J/cm2 LLLI. Specific Real-Time PCR primers were designed to amplify all the two CXCR4 isoforms yet identified. RNA amplifications were done for all the groups. We showed for the first time that LLLI makes the human blastocysts to increase the expression level of CXCR4 a few hours after irradiation. Moreover, it was shown that two irradiation doses with one day interval can cause a significant increase in CXCR4 expression level in human blastocysts. This study revealed that LLLI could be a proliferation motivator for embryonic cell divisions through enhanced over-expression of CXCR4 level.

Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

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A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system ...

Heterologous expression of a rice metallothionein isoform (OsMTI-1b in Saccharomyces cerevisiae enhances cadmium, hydrogen peroxide and ethanol tolerance

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Zahra Ansarypour

Full Text Available Abstract Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b on the tolerance of Saccharomyces cerevisiae to Cd2+, H2O2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd2+ and accumulated more Cd2+ ions when they were grown in the medium containing CdCl2. In addition, the heterologous expression of GST-OsMTI-1b conferred H2O2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses.

Expression of DAI by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity

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Mari Hirvinen

2016-01-01

Full Text Available In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate—and eventually the long-lasting adaptive immunity against cancer.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

Science.gov (United States)

Matsumoto, M; Imagawa, M; Aoki, Y

2000-07-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.

Transforming growth factor-β inhibits CCAAT/enhancer-binding protein expression and PPARγ activity in unloaded bone marrow stromal cells

International Nuclear Information System (INIS)

Ahdjoudj, S.; Kaabeche, K.; Holy, X.; Fromigue, O.; Modrowski, D.; Zerath, E.; Marie, P.J.

2005-01-01

The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-β2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP)α and C/EBPβ α at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor γ (PPARγ2) transcripts at 7 days. TGF-β2 administration in unloaded rats corrected the rise in C/EBPα and C/EBPβ transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPARγ2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBPα and C/EBPβ expression by TGF-β2 was associated with increased PPARγ serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPARγ transactivating activity. The sequential inhibitory effect of TGF-β2 on C/EBPα, C/EBPβ, and PPARγ2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-β2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBPα, C/EBPβ, and PPARγ expression and activity, which provides a sequential mechanism by which TGF-β2 regulates adipogenic differentiation of bone marrow stromal cells in vivo

Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

International Nuclear Information System (INIS)

Miyatake, Kazumasa; Tsuji, Kunikazu; Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer; Sekiya, Ichiro; Muneta, Takeshi

2013-01-01

Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding the molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis

Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

Energy Technology Data Exchange (ETDEWEB)

Miyatake, Kazumasa [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Tsuji, Kunikazu, E-mail: ktsuji.gcoe@tmd.ac.jp [International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan); Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Sekiya, Ichiro [Section of Cartilage Regeneration, Tokyo Medical and Dental University, Tokyo (Japan); Muneta, Takeshi [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan)

2013-02-01

Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding the molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis.

Triple-Gene Therapy for Stroke: A Proof-of-Concept in Vivo Study in Rats

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Mikhail E. Sokolov

2018-02-01

Full Text Available Natural brain repair after stroke is extremely limited, and current therapeutic options are even more scarce with no clinical break-through in sight. Despite restricted regeneration in the central nervous system, we have previously proved that human umbilical cord blood mono-nuclear cells (UCB-MC transduced with adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF, glial cell-derived neurotrophic factor (GDNF, and neural cell adhesion molecule (NCAM successfully rescued neurons in amyotrophic lateral sclerosis and spinal cord injury. This proof-of-principle project was aimed at evaluating the beneficial effects of the same triple-gene approach in stroke. Rats subjected to distal occlusion of the middle cerebral artery were treated intrathecally with a combination of these genes either directly or using our cell-based (UCB-MC approach. Various techniques and markers were employed to evaluate brain injury and subsequent recovery after treatment. Brain repair was most prominent when therapeutic genes were delivered via adenoviral vector- or UCB-MC-mediated approach. Remodeling of brain cortex in the stroke area was confirmed by reduction of infarct volume and attenuated neural cell death, depletion of astrocytes and microglial cells, and increase in the number of oligodendroglial cells and synaptic proteins expression. These results imply that intrathecal injection of genetically engineered UCB-MC over-expressing therapeutic molecules (VEGF, GDNF, and NCAM following cerebral blood vessel occlusion might represent a novel avenue for future research into treating stroke.

Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

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Besson, Marie Thérèse; Alegría, Karin; Garrido-Gerter, Pamela; Barros, Luis Felipe; Liévens, Jean-Charles

2015-01-01

Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93). We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP) impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK) which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to mediate the hGluT3

Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

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Marie Thérèse Besson

Full Text Available Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93. We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD, the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to

Role of Estrogens in the Size of Neuronal Somata of Paravaginal Ganglia in Ovariectomized Rabbits

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Laura G. Hernández-Aragón

2017-01-01

Full Text Available We aimed to determine the role of estrogens in modulating the size of neuronal somata of paravaginal ganglia. Rabbits were allocated into control (C, ovariectomized (OVX, and OVX treated with estradiol benzoate (OVX + EB groups to evaluate the neuronal soma area; total serum estradiol (E2 and testosterone (T levels; the percentage of immunoreactive (ir neurons anti-aromatase, anti-estrogen receptor (ERα, ERβ and anti-androgen receptor (AR; the intensity of the immunostaining anti-glial cell line-derived neurotrophic factor (GDNF and the GDNF family receptor alpha type 1 (GFRα1; and the number of satellite glial cells (SGCs per neuron. There was a decrease in the neuronal soma size for the OVX group, which was associated with low T, high percentages of aromatase-ir and neuritic AR-ir neurons, and a strong immunostaining anti-GDNF and anti-GFRα1. The decrease in the neuronal soma size was prevented by the EB treatment that increased the E2 without affecting the T levels. Moreover, there was a high percentage of neuritic AR-ir neurons, a strong GDNF immunostaining in the SGC, and an increase in the SGCs per neuron. Present findings show that estrogens modulate the soma size of neurons of the paravaginal ganglia, likely involving the participation of the SGC.

Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

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Mädje Katharina

2012-01-01

Full Text Available Abstract Background Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(PH. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH, presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S-1-(2-chlorophenylethanol from o-chloroacetophenone. Results As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3 that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by

Enhanced cerebrovascular expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 via the MEK/ERK pathway during cerebral ischemia in the rat

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Maddahi Aida

2009-06-01

Full Text Available Abstract Background Cerebral ischemia is usually characterized by a reduction in local blood flow and metabolism and by disruption of the blood-brain barrier in the infarct region. The formation of oedema and opening of the blood-brain barrier in stroke is associated with enhanced expression of metalloproteinase-9 (MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1. Results Here, we found an infarct volume of 24.8 ± 2% and a reduced neurological function after two hours of middle cerebral artery occlusion (MCAO, followed by 48 hours of recirculation in rat. Immunocytochemistry and confocal microscopy revealed enhanced expression of MMP-9, TIMP-1, and phosphorylated ERK1/2 in the smooth muscle cells of the ischemic MCA and associated intracerebral microvessels. The specific MEK1/2 inhibitor U0126, given intraperitoneal zero or 6 hours after the ischemic event, reduced the infarct volume significantly (11.8 ± 2% and 14.6 ± 3%, respectively; P Conclusion These data are the first to show that the elevated vascular expression of MMP-9 and TIMP-1, associated with breakdown of the blood-brain barrier following focal ischemia, are transcriptionally regulated via the MEK/ERK pathway.

Assisted Reproduction Causes Reduced Fetal Growth Associated with Downregulation of Paternally Expressed Imprinted Genes That Enhance Fetal Growth in Mice.

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Li, Bo; Chen, Shuqiang; Tang, Na; Xiao, Xifeng; Huang, Jianlei; Jiang, Feng; Huang, Xiuying; Sun, Fangzhen; Wang, Xiaohong

2016-02-01

Alteration of intrauterine growth trajectory is linked to metabolic diseases in adulthood. In mammalian and, specifically, human species, pregnancies through assisted reproductive technology (ART) are associated with changes in intrauterine growth trajectory. However, it is still unclear how ART alters intrauterine growth trajectory, especially reduced fetal growth in early to midgestation. In this study, using a mouse model, it was found that ART procedures reduce fetal and placental growth at Embryonic Day 10.5. Furthermore, ART leads to decreased methylation levels at H19, KvDMR1, and Snrpn imprinting control regions in the placentae, instead of fetuses. Furthermore, in the placenta, ART downregulated a majority of parentally expressed imprinted genes, which enhance fetal growth, whereas it upregulated a majority of maternally expressed genes which repress fetal growth. Additionally, the expression of genes that regulate placental development was also affected by ART. ART also downregulated a majority of placental nutrient transporters. Disruption of genomic imprinting and abnormal expression of developmentally and functionally relevant genes in placenta may influence the placental development and function, which affect fetal growth and reprogramming. © 2016 by the Society for the Study of Reproduction, Inc.

Oxide Synthase Expression by p38 MAP Kinase

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Tuija Turpeinen

2011-01-01

Full Text Available The role of dual specificity phosphatase 1 (DUSP1 in inducible nitric oxide synthase (iNOS expression in A549 human pulmonary epithelial cells, J774 mouse macrophages and primary mouse bone marrow-derived macrophages (BMMs was investigated. iNOS expression was induced by a cytokine mixture (TNF, IFNγ and IL-1β in A549 cells and by LPS in J774 cells, and it was inhibited by p38 MAPK inhibitors SB202190 and BIRB 796. Stimulation with cytokine mixture or LPS enhanced also DUSP1 expression. Down-regulation of DUSP1 by siRNA increased p38 MAPK phosphorylation and iNOS expression in A549 and J774 cells. In addition, LPS-induced iNOS expression was enhanced in BMMs from DUSP1(−/− mice as compared to that in BMMs from wild-type mice. The results indicate that DUSP1 suppresses iNOS expression by limiting p38 MAPK activity in human and mouse cells. Compounds that enhance DUSP1 expression or modulate its function may be beneficial in diseases complicated with increased iNOS-mediated NO production.

Neuroprotective and anti-inflammatory mechanisms are activated early in optic neuritis

DEFF Research Database (Denmark)

Tsakiri, A; Ravanidis, S; Lagoudaki, R

2015-01-01

-matched voluntary HC (n = 32). The mRNA expression of distinct cytokines and neurotrophic factors was assessed by semi/quantitative real-time PCR (RT-PCR). RESULTS: Brain- and glial cell-derived neurotrophic factor (BDNF and GDNF) and interleukin 10 (IL-10) expression was significantly increased in the CSF compared......). METHODS: Sixty-four patients between 18 and 59 years of age suffering by acute ON, onset of brain magnetic resonance imaging (MRI) were performed in ON. Blood and CSF samples were collected from untreated patients and from a gender- and age...... to the blood in both ON and HC (P brain MRI showed increased levels of IL-5 in blood (P = 0.03). CONCLUSION: Our...

Expression of TpNRAMP5, a metal transporter from Polish wheat (Triticum polonicum L.), enhances the accumulation of Cd, Co and Mn in transgenic Arabidopsis plants.

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Peng, Fan; Wang, Chao; Zhu, Jianshu; Zeng, Jian; Kang, Houyang; Fan, Xing; Sha, Lina; Zhang, Haiqin; Zhou, Yonghong; Wang, Yi

2018-06-01

TpRNAMP5 is mainly expressed in the plasma membrane of roots and basal stems. It functions as a metal transporter for Cd, Mn and Co accumulation. Numerous natural resistance-associated macrophage proteins (NRAMPs) have been functionally identified in various plant species, including Arabidopsis, rice, soybean and tobacco, but no information is available on NRAMP genes in wheat. In this study, we isolated a TpNRAMP5 from dwarf Polish wheat (DPW, Triticum polonicum L.), a species with high tolerance to Cd and Zn. Expression pattern analysis revealed that TpNRAMP5 is mainly expressed in roots and basal stems of DPW. TpNRAMP5 was localized at the plasma membrane of Arabidopsis leaf protoplast. Expression of TpNRAMP5 in yeast significantly increased yeast sensitivity to Cd and Co, but not Zn, and enhanced Cd and Co concentrations. Expression of TpNRAMP5 in Arabidopsis significantly increased Cd, Co and Mn concentrations in roots, shoots and whole plants, but had no effect on Fe and Zn concentrations. These results indicate that TpNRAMP5 is a metal transporter enhancing the accumulation of Cd, Co and Mn, but not Zn and Fe. Genetic manipulation of TpNRAMP5 can be applied in the future to limit the transfer of Cd from soil to wheat grains, thereby protecting human health.

Combination effects of epidermal growth factor and glial cell line-derived neurotrophic factor on the in vitro developmental potential of porcine oocytes

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Valleh, Mehdi Vafaye; Rasmussen, Mikkel Aabech; Hyttel, Poul

2016-01-01

of improving this issue, the single and combined effects of epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) on oocyte developmental competence were investigated. Porcine cumulus–oocyte cell complexes (COCs) were matured in serum-free medium supplemented with EGF (0, 10...... with the combination of EGF and GDNF was shown to significantly improve oocyte competence in terms of blastocyst formation, blastocyst cell number and blastocyst hatching rate (P

Expression of chickpea CIPK25 enhances root growth and tolerance to dehydration and salt stress in transgenic tobacco

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Mukesh Kumar Meena

2015-09-01

Full Text Available Calcium signaling plays an important role in adaptation and developmental processes in plants and animals. A class of calcium sensors, known as Calcineurin B-like (CBL proteins sense specific temporal changes in cytosolic Ca2+ concentration and regulate activities of a group of ser/thr protein kinases called CBL-interacting protein kinases (CIPKs. Although a number of CIPKs have been shown to play crucial roles in the regulation of stress signaling, no study on the function of CIPK25 or its orthologues has been reported so far. In the present study, an orthologue of Arabidopsis CIPK25 was cloned from chickpea (Cicer arietinum. CaCIPK25 gene expression in chickpea increased upon salt, dehydration, and different hormonal treatments. CaCIPK25 gene showed differential tissue-specific expression. 5'-upstream activation sequence (5'-UAS of the gene and its different truncated versions were fused to a reporter gene and studied in Arabidopsis to identify promoter regions directing its tissue-specific expression. Replacement of a conserved threonine residue with an aspartic acid at its catalytic site increased the kinase activity of CaCIPK25 by 2.5-fold. Transgenic tobacco plants overexpressing full-length and the high active versions of CaCIPK25 displayed a differential germination period and longer root length in comparison to the control plants. Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions. Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants. Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

7-ketocholesteryl-9-carboxynonanoate enhances ATP binding cassette transporter A1 expression mediated by PPARγ in THP-1 macrophages.

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Chi, Yan; Wang, Le; Liu, Yuanyuan; Ma, Yanhua; Wang, Renjun; Han, Xiaofei; Qiao, Hui; Lin, Jiabin; Matsuura, Eiji; Liu, Shuqian; Liu, Qingping

2014-06-01

ATP binding cassette transporter A1 (ABCA1) is a member of the ATP-binding cassette transporter family. It plays an essential role in mediating the efflux of excess cholesterol. It is known that peroxisome proliferator-activated receptor gamma (PPARγ) promoted ABCA1 expression. We previously found 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) upregulated ABCA1 partially through CD36 mediated signals. In the present study, we intended to test if PPARγ signally is involved in the upregulation mediated by oxLig-1. First, we docked oxLig-1 and the ligand-binding domain (LBD) of PPARγ by using AutoDock 3.05 and subsequently confirmed the binding by ELISA assay. Western blotting analyses showed that oxLig-1 induces liver X receptor alpha (LXRα), PPARγ and consequently ABCA1 expression. Furthermore, oxLig-1 significantly enhanced ApoA-I-mediated cholesterol efflux. Pretreatment with an inhibitor for PPARγ (GW9662) or/and LXRα (GGPP) attenuated oxLig-1-induced ABCA1 expression. Under PPARγ knockdown by using PPARγ-shRNA, oxLig-1-induced ABCA1 expression and cholesterol efflux in THP-1 macrophages was blocked by 62% and 25% respectively. These observations suggest that oxLig-1 is a novel PPARγ agonist, promoting ApoA-I-mediated cholesterol efflux from THP-1 macrophages by increasing ABCA1 expression via induction of PPARγ. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

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Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

2016-04-01

Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. Copyright © 2016 Elsevier Inc. All rights reserved.

Tet1 overexpression leads to anxiety-like behavior and enhanced fear memories via the activation of calcium-dependent cascade through Egr1 expression in mice.

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Kwon, Wookbong; Kim, Hyeng-Soo; Jeong, Jain; Sung, Yonghun; Choi, Minjee; Park, Song; Lee, Jinhee; Jang, Soyoung; Kim, Sung Hyun; Lee, Sanggyu; Kim, Myoung Ok; Ryoo, Zae Young

2018-01-01

Ten-eleven translocation methylcytosine dioxygenase 1 ( Tet1 ) initiates DNA demethylation by converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) at CpG-rich regions of genes, which have key roles in adult neurogenesis and memory. In addition, the overexpression of Tet1 with 5-hmC alteration in patients with psychosis has also been reported, for instance in schizophrenia and bipolar disorders. The mechanism underlying Tet1 overexpression in the brain; however, is still elusive. In the present study, we found that Tet1-transgenic (Tet1-TG) mice displayed abnormal behaviors involving elevated anxiety and enhanced fear memories. We confirmed that Tet1 overexpression affected adult neurogenesis with oligodendrocyte differentiation in the hippocampal dentate gyrus of Tet1-TG mice. In addition, Tet1 overexpression induced the elevated expression of immediate early genes, such as Egr1 , c-fos , Arc , and Bdnf , followed by the activation of intracellular calcium signals ( i.e. , CamKII, ERK, and CREB) in prefrontal and hippocampal neurons. The expression of GABA receptor subunits ( Gabra2 and Gabra4 ) fluctuated in the prefrontal cortex and hippocampus. We evaluated the effects of Tet1 overexpression on intracellular calcium-dependent cascades by activating the Egr1 promoter in vitro Tet1 enhanced Egr1 expression, which may have led to alterations in Gabra2 and Gabra4 expression in neurons. Taken together, we suggest that the Tet1 overexpression in our Tet1-TG mice can be applied as an effective model for studying various stress-related diseases that show hyperactivation of intracellular calcium-dependent cascades in the brain.-Kwon, W., Kim, H.-S., Jeong, J., Sung, Y., Choi, M., Park, S., Lee, J., Jang, S., Kim, S. H., Lee, S., Kim, M. O., Ryoo, Z. Y. Tet1 overexpression leads to anxiety-like behavior and enhanced fear memories via the activation of calcium-dependent cascade through Egr1 expression in mice. © FASEB.

Compartmentalised expression of meprin in small intestinal mucosa: enhanced expression in lamina propria in coeliac disease.

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Lottaz, Daniel; Buri, Caroline; Monteleone, Giovanni; Rösmann, Sandra; Macdonald, Thomas T; Sanderson, Ian R; Sterchi, Erwin E

2007-03-01

Epithelial cells in the human small intestine express meprin, an astacin-like metalloprotease, which accumulates normally at the brush border membrane and in the gut lumen. Therefore, meprin is targeted towards luminal components. In coeliac disease patients, peptides from ingested cereals trigger mucosal inflammation in the small intestine, disrupting epithelial cell differentiation and function. Using in situ hybridisation on duodenal tissue sections, we observed a marked shift of meprin mRNA expression from epithelial cells, the predominant expression site in normal mucosa, to lamina propria leukocytes in coeliac disease. Meprin thereby gains access to the substrate repertoire present beneath the epithelium.

Motif signatures of transcribed enhancers

KAUST Repository

Kleftogiannis, Dimitrios A.; Ashoor, Haitham; Zarokanellos, Nikolaos; Bajic, Vladimir B.

2017-01-01

In mammalian cells, transcribed enhancers (TrEn) play important roles in the initiation of gene expression and maintenance of gene expression levels in spatiotemporal manner. One of the most challenging questions in biology today is how the genomic

Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles

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Sugino, Noriko [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Miura, Yasuo, E-mail: ym58f5@kuhp.kyoto-u.ac.jp [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Yao, Hisayuki [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Iwasa, Masaki; Fujishiro, Aya [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Division of Gastroenterology and Hematology, Shiga University of Medical Science, Shiga 520-2192 (Japan); Fujii, Sumie [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Hirai, Hideyo [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Takaori-Kondo, Akifumi [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Ichinohe, Tatsuo [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Maekawa, Taira [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan)

2016-01-22

Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34{sup +} hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation. - Highlights: • Human BM-MSCs in an early osteoinductive stage (e-MSCs) support hematopoiesis. • Adhesion- and chemotaxis-associated gene signatures are altered in e-MSCs. • Expression of CXCL12 and VCAM1 is remarkably decreased in e-MSCs. • e-MSCs are at differentiation stage between MSCs and osteoprogenitor cells. • Osteoinductive treatment

Exercise improves cognitive responses to psychological stress through enhancement of epigenetic mechanisms and gene expression in the dentate gyrus.

Directory of Open Access Journals (Sweden)

Andrew Collins

Full Text Available We have shown previously that exercise benefits stress resistance and stress coping capabilities. Furthermore, we reported recently that epigenetic changes related to gene transcription are involved in memory formation of stressful events. In view of the enhanced coping capabilities in exercised subjects we investigated epigenetic, gene expression and behavioral changes in 4-weeks voluntarily exercised rats.Exercised and control rats coped differently when exposed to a novel environment. Whereas the control rats explored the new cage for the complete 30-min period, exercised animals only did so during the first 15 min after which they returned to sleeping or resting behavior. Both groups of animals showed similar behavioral responses in the initial forced swim session. When re-tested 24 h later however the exercised rats showed significantly more immobility behavior and less struggling and swimming. If rats were killed at 2 h after novelty or the initial swim test, i.e. at the peak of histone H3 phospho-acetylation and c-Fos induction, then the exercised rats showed a significantly higher number of dentate granule neurons expressing the histone modifications and immediate-early gene induction.Thus, irrespective of the behavioral response in the novel cage or initial forced swim session, the impact of the event at the dentate gyrus level was greater in exercised rats than in control animals. Furthermore, in view of our concept that the neuronal response in the dentate gyrus after forced swimming is involved in memory formation of the stressful event, the observations in exercised rats of enhanced neuronal responses as well as higher immobility responses in the re-test are consistent with the reportedly improved cognitive performance in these animals. Thus, improved stress coping in exercised subjects seems to involve enhanced cognitive capabilities possibly resulting from distinct epigenetic mechanisms in dentate gyrus neurons.

UV-enhanced reactivation in mammalian cells: increase by caffeine

International Nuclear Information System (INIS)

Lytle, C.D.; Iacangelo, A.L.; Lin, C.H.; Goddard, J.G.

1981-01-01

It has been reported that caffeine decreases UV-enhanced reactivation of UV-irradiated Herpes simplex virus in CV-l monkey kidney cells. That occurred when there was no delay between cell irradiation and virus infection. In the present study, virus infection was delayed following cell irradiation to allow an 'induction' period separate from the 'expression' period which occurs during the virus infection. Thus, the effects of caffeine on 'induction' and 'expression' could be determined separately. Caffeine increased the expression of UV-enhanced reactivation, while causing a small decrease in the 'induction' of enhanced reactivation. (author)

Increased p53 and decreased p21 accompany apoptosis induced by ultraviolet radiation in the nervous system of a crustacean

International Nuclear Information System (INIS)

Hollmann, Gabriela; Linden, Rafael; Giangrande, Angela; Allodi, Silvana

2016-01-01

Highlights: • The paper characterizes molecular pathways of cell responses to environmental doses of UV in brain tissue of a crab species. • The UV radiation changes levels of proteins which trigger apoptotic or cell cycle arrest pathways and also it changes neurotrophins which lead to apoptosis of neural cell in the central nervous system (CNS) of the crab Ucides cordatus. • The UVB wavelengths in the solar simulator damaged the DNA, either directly or indirectly, by increasing ROS, and induced the increase of p53 and AKT, which blocked p21 and increased the expression of activated caspase-3, triggering apoptosis. The signs of death increased the expression of neurotrophins (BDNF and GDNF), which continued to stimulate the apoptosis signaling mediated by caspase-3. • In the brain of the crab U. cordatus, p53/p21 relationship in response to UV radiation is different from that of most mammals. - Abstract: Ultraviolet (UV) radiation can produce biological damage, leading the cell to apoptosis by the p53 pathway. This study evaluated some molecular markers of the apoptosis pathway induced by UVA, UVB and UVA+ UVB (Solar Simulator, SIM) in environmental doses, during five consecutive days of exposure, in the brain of the crab Ucides cordatus. We evaluated the central nervous system (CNS) by immunoblotting the content of proteins p53, p21, phosphorylated AKT, BDNF, GDNF, activated caspase-3 (C3) and phosphohistone H3 (PH3); and by immunohistochemical tests of the cells labeled for PH3 and C3. After the fifth day of exposure, UVB radiation and SIM increased the protein content of p53, increasing the content of AKT and, somehow, blocking p21, increasing the content of activated caspase-3, which led the cells to apoptosis. The signs of death affected the increase in neurotrophins, such as BDNF and GDNF, stimulating the apoptotic cascade of events. Immunohistochemical assays and immunoblotting showed that apoptosis was present in the brains of all UV groups, while

Increased p53 and decreased p21 accompany apoptosis induced by ultraviolet radiation in the nervous system of a crustacean

Energy Technology Data Exchange (ETDEWEB)

Hollmann, Gabriela, E-mail: gabrielahollmann@biof.ufrj.br [Programa de Pós Graduação em Ciências Biológicas-Fisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ 21941-590 (Brazil); Linden, Rafael, E-mail: rlinden@biof.ufrj.br [Programa de Pós Graduação em Ciências Biológicas-Fisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ 21941-590 (Brazil); Giangrande, Angela, E-mail: angela.giangrande@igbmc.fr [Institut de Génétique et de Biologie Moléculaire et Cellulaire-IGBMC, INSERM, Strasbourg (France); Allodi, Silvana, E-mail: sallodi@biof.ufrj.br [Programa de Pós Graduação em Ciências Biológicas-Fisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ 21941-590 (Brazil)

2016-04-15

Highlights: • The paper characterizes molecular pathways of cell responses to environmental doses of UV in brain tissue of a crab species. • The UV radiation changes levels of proteins which trigger apoptotic or cell cycle arrest pathways and also it changes neurotrophins which lead to apoptosis of neural cell in the central nervous system (CNS) of the crab Ucides cordatus. • The UVB wavelengths in the solar simulator damaged the DNA, either directly or indirectly, by increasing ROS, and induced the increase of p53 and AKT, which blocked p21 and increased the expression of activated caspase-3, triggering apoptosis. The signs of death increased the expression of neurotrophins (BDNF and GDNF), which continued to stimulate the apoptosis signaling mediated by caspase-3. • In the brain of the crab U. cordatus, p53/p21 relationship in response to UV radiation is different from that of most mammals. - Abstract: Ultraviolet (UV) radiation can produce biological damage, leading the cell to apoptosis by the p53 pathway. This study evaluated some molecular markers of the apoptosis pathway induced by UVA, UVB and UVA+ UVB (Solar Simulator, SIM) in environmental doses, during five consecutive days of exposure, in the brain of the crab Ucides cordatus. We evaluated the central nervous system (CNS) by immunoblotting the content of proteins p53, p21, phosphorylated AKT, BDNF, GDNF, activated caspase-3 (C3) and phosphohistone H3 (PH3); and by immunohistochemical tests of the cells labeled for PH3 and C3. After the fifth day of exposure, UVB radiation and SIM increased the protein content of p53, increasing the content of AKT and, somehow, blocking p21, increasing the content of activated caspase-3, which led the cells to apoptosis. The signs of death affected the increase in neurotrophins, such as BDNF and GDNF, stimulating the apoptotic cascade of events. Immunohistochemical assays and immunoblotting showed that apoptosis was present in the brains of all UV groups, while

Self-expression assignment as a teaching approach to enhance the interest of Kuwaiti women in biological sciences.

Science.gov (United States)

El-Sabban, Farouk

2008-06-01

Stimulating the interest of students in biological sciences necessitates the use of new teaching methods and motivating approaches. The idea of the self-expression assignment (SEA) has evolved from the prevalent environment at the College for Women of Kuwait University (Safat, State of Kuwait), a newly established college where the number of students is low and where students have varied backgrounds and interests and are being instructed biological sciences in English for the first time. This SEA requires each student to choose a topic among a long list of topics and interact with it in any way to produce a finished product without the interference of the course instructor. Students are told that the SEA will be graded based on their commitment, creative thinking, innovation in developing the idea, and finishing up of the chosen assignment. The SEA has been implemented in three introductory courses, namely, Biology, Introduction to Human Nutrition and Food Science, and The Human Body. Many interesting projects resulted from the SEA, and, based on an administered survey, students assessed this assignment very favorably. Students expressed their pleasure of experiencing freedom in choosing their own topics, interacting with such topics, learning more about them, and finishing up their projects. Students appreciated this type of exposure to biological sciences and expressed that such an experience enhanced their interest in such sciences.

The defense-responsive genes showing enhanced and repressed expression after pathogen infection in rice (Oryza sativa L.)

Institute of Scientific and Technical Information of China (English)

ZHOU; Bin(周斌); PENG; Kaiman(彭开蔓); CHU; Zhaohui(储昭晖); WANG; Shiping(王石平); ZHANG; Qifa(张启发)

2002-01-01

Despite large numbers of studies about defense response, processes involved in the resistance of plants to incompatible pathogens are still largely uncharacterized. The objective of this study was to identify genes involved in defense response by cDNA array analysis and to gain knowledge about the functions of the genes involved in defense response. Approximately 20000 rice cDNA clones were arrayed on nylon filters. RNA samples isolated from different rice lines after infection with incompatible strains or isolates of Xanthomonas oryzae pv. oryzae or Pyricularia grisea, respectively, were used to synthesize cDNA as probes for screening the cDNA arrays. A total of 100 differentially expressed unique sequences were identified from 5 pathogen-host combinations. Fifty-three sequences were detected as showing enhanced expression and 47 sequences were detected as showing repressed expression after pathogen infection. Sequence analysis revealed that most of the 100 sequences had various degrees of homology with genes in databases which encode or putatively encode transcription regulating proteins, translation regulating proteins, transport proteins, kinases, metabolic enzymes, and proteins involved in other functions. Most of the genes have not been previously reported as being involved in the disease resistance response in rice. The results from cDNA arrays, reverse transcription-polymerase chain reaction, and RNA gel blot analysis suggest that activation or repression of most of these genes might occur commonly in the defense response.

Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration

International Nuclear Information System (INIS)

Xiao Caiwen; Zhou Huifang; Fu Yao; Gu Ping; Fan Xianqun; Liu Guangpeng; Zhang Peng; Hou Hongliang; Tang Tingting

2011-01-01

Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.