Empty Control Plasmid Research Articles

The RRE-REV module has no effect on the packaging efficiency of cas9 and Gag proteins into nanomedic virus-like particles

Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing, suitable for gene therapy of human diseases in the future. Efficiency of genome editing with VLPs depends on the packaging of Cas9 into VLPs, that is mediated by viral Gag protein. To increase the packaging of Cas9 into NanoMEDIC system VLPs plasmid constructs for expression of Cas9 and Gag were modified by the addition of HIV RRE (Rev response element), that is expected to increase the nuclear export of RRE-containing transcripts to cytosol via accessory protein Rev, as described for Vpr-Cas9-based VLP system. Here we found that Cas9 and Gag protein levels in the cell lysates are increased upon cotransfection of either Rev-expressing plasmid or empty control plasmid. Moreover, this effect does not depend on the presence of RRE in the transcript. On the top of that, we showed that AP21967-induced dimerization of FRB and FKBP12, but not the modification of plasmids with RRE and/or cotransfection of Rev-expressing plasmid, plays the major role in packaging of Cas9 into NanoMEDIC system VLPs. These data suggest that it is impractical to use the RRE-Rev module to enhance the packing of Cas9 nuclease into VLPs.

The RRE-Rev Module Has No Effect on the Packaging Efficiency of Cas9 and Gag Proteins into NanoMEDIC Virus-like Particles.

Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing and is suitable for gene therapy of human diseases in the future. The efficiency of genome editing with VLPs depends on the Cas9 packaging into VLPs, the process mediated by the viral Gag protein. To improve the packaging of Cas9 into NanoMEDIC VLPs, plasmid constructs for Cas9 and Gag expression were modified by adding the HIV Rev response element (RRE), which was expected to increase the nuclear export of RRE-containing transcripts into the cytosol via the Rev accessory protein, as described for a Vpr-Cas9-based VLP system. The Cas9 and Gag protein levels in cell lysates were found to increase upon cotransfection with either the Rev-expressing plasmid or the empty control plasmid. The effect was independent of the presence of RRE in the transcript. Moreover, AP21967-induced dimerization of FRB and FKBP12, but not plasmid modification with RRE and/or cotransfection with the Rev-expressing plasmid, was shown to play the major role in Cas9 packaging into NanoMEDIC VLPs. The data indicated that it is impractical to use the RRE-Rev module to enhance the packaging of Cas9 nuclease into VLPs.

Identification of Abnormal Spindle Microtubule Assembly as a Promising Therapeutic Target for Osteosarcoma.

ObjectiveTo demonstrate the expression of abnormal spindle microtubule assembly (ASPM) in clinical osteosarcoma tissue specimens collected in our hospital, and to explore the function of ASPM in osteosarcoma in vitro and in vivo.MethodsTissue specimens from 82 cases of osteosarcoma were collected and analyzed by immunohistochemistry assay. We also investigated the relationship between ASPM expression and clinicopathological characteristics in the patients. We transfected shASPM plasmid and the empty control plasmid, respectively, and then used quantitative polymerase chain reaction and western blot analysis to detect ASPM expression. Cell colony assay and MTT were used to observe the proliferation ability. In vivo study was undertaken to explore the ASPM function further.ResultsIn this study, ASPM showed high expression in osteosarcoma tissue samples compared with non‐tumor normal tissues. ASPM was positively correlated with clinical pathological characteristics, including tumor size (P = 0.024) and clinical stage (P = 0.045). Our results further showed that ASPM depletion dramatically inhibited the proliferation of osteosarcoma cells (with fewer cells in the sh‐RNA‐ASPM group compared with the control group(P < 0.05, respectively), and the in vivo assays further confirmed that ASPM ablation markedly blocked tumor growth compared with control (P < 0.05).ConclusionOur data provides strong evidence that the high expression of ASPM in osteosarcoma promotes proliferation in vitro and in vivo, indicating its potential role as an osteosarcoma therapeutic target.

Repression of Death Receptor–Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is associated with viral persistence and pathogenesis. Resistance of HBV-infected hepatocytes to apoptosis is seen as one of the primary promotors for HBV chronicity and malignancy. Fas receptor/ligand (Fas/FasL) and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) system plays a key role in hepatic death during HBV infection. We found that HBeAg mediates resistance of hepatocytes to FasL or TRAIL-induced apoptosis. Introduction of HBeAg into human hepatocytes rendered resistance to FasL or TRAIL cytotoxicity in a p53-dependent manner. HBeAg further inhibited the expression of p53, total Fas, membrane-bound Fas, TNF receptor superfamily member 10a, and TNF receptor superfamily member 10b at both mRNA and protein levels. Incontrast, HBeAg enhanced the expression of soluble forms of Fas through facilitation of Fas alternative mRNA splicing. In a mouse model, expression of HBeAg in mice injected with recombinant adenovirus-associated virus 8 inhibited agonistic anti-Fas antibody-induced hepatic apoptosis. Xenograft tumorigenicity assay also found that HBeAg-induced carcinogenesis was resistant to the proapoptotic effect of TRAIL and chemotherapeutic drugs. These results indicate that HBeAg may prevent hepatocytes from FasL and TRAIL-induced apoptosis by regulating the expression of the proapoptotic and antiapoptotic forms of death receptors, which may contribute to the survival and persistence of infected hepatocytes during HBV infection.

The evaluation of fibrotic effects of the hepatitis B virus pre-core in hepatic stellate cells.

The role of the hepatitis B virus (HBV) endogenous pre-core protein in liver fibrosis is controversial. Whether the expression of the pre-core induces the activation of human stellate cells (HSCs) has not yet been reported. Plasmids expressing HBx, or pre-core protein were transfected into LX-2 cells. Subsequently, total RNA extracted and reverse transcription-quantitative polymerase chain reaction was performed to measure the fold change of collagen type I, α1 chain, α-smooth muscle actin and TIMP metalloproteinase inhibitor-1. Moreover, transforming growth factor (TGF)-β in the supernatant of HSCs was evaluated by ELISA assay. In addition, a MTT assay was performed to test the cytotoxicity of the endogenous expression in LX-2 cells. None of the plasmids exhibited cytotoxic nor significant proliferative effects on LX-2 cells by MTT assessment. The gene expression analysis of fibrotic genes in LX-2 cells demonstrated that the pre-core protein presented no significant (P>0.05) fibrotic impact when compared to the empty control plasmid and HBx. The data from the TGF-β ELISA was consistent with the mRNA expression as detected with the control plasmid (P>0.05). The endogenous expression of the HBV pre-core exhibited no fibrotic impression in HSCs when compared to HBx.

Decreased capacity for sodium export out of Arabidopsis chloroplasts impairs salt tolerance, photosynthesis and plant performance

Salt stress is a widespread phenomenon, limiting plant performance in large areas around the world. Although various types of plant sodium/proton antiporters have been characterized, the physiological function of NHD1 from Arabidopsis thaliana has not been elucidated in detail so far. Here we report that the NHD1-GFP fusion protein localizes to the chloroplast envelope. Heterologous expression of AtNHD1 was sufficient to complement a salt-sensitive Escherichia coli mutant lacking its endogenous sodium/proton exchangers. Transport competence of NHD1 was confirmed using recombinant, highly purified carrier protein reconstituted into proteoliposomes, proving Na(+) /H(+) antiport. In planta NHD1 expression was found to be highest in mature and senescent leaves but was not induced by sodium chloride application. When compared to wild-type controls, nhd1 T-DNA insertion mutants showed decreased biomasses and lower chlorophyll levels after sodium feeding. Interestingly, if grown on sand and supplemented with high sodium chloride, nhd1 mutants exhibited leaf tissue Na(+) levels similar to those of wild-type plants, but the Na(+) content of chloroplasts increased significantly. These high sodium levels in mutant chloroplasts resulted in markedly impaired photosynthetic performance as revealed by a lower quantum yield of photosystem II and increased non-photochemical quenching. Moreover, high Na(+) levels might hamper activity of the plastidic bile acid/sodium symporter family protein 2 (BASS2). The resulting pyruvate deficiency might cause the observed decreased phenylalanine levels in the nhd1 mutants due to lack of precursors.

Distinct Modes of Inhibition by Sclerostin on Bone Morphogenetic Protein and Wnt Signaling Pathways

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.

Hyperthermia-induced apoptosis in Tca8113 cells is inhibited by heat shock protein 27 through blocking phospholipid scramblase 3 phosphorylation

Purpose: Hyperthermia induces tumour cell apoptosis through the mitochondrial apoptotic pathway; however, the signal transduction mechanism underlying this process still needs to be fully elucidated. Phospholipid scramblase 3 (PLS3), a target of protein kinase C-δ (PKC-δ), resides in mitochondria and plays pivotal roles in regulating apoptotic response. Activated PLS3 facilitates cardiolipin (CL) translocation from the mitochondrial inner membrane to the outer leaflet of the mitochondrial outer membrane and triggers apoptosis.Materials and methods: The tongue squamous cell carcinoma Tca8113 cells were transfected or co-transfected using Lipofectamine 2000 with plasmids pCMV-6×His-PLS3, pCMV-6×His-PLS3 (T21A), pHA-PKC-δ, pHA-PKC-δ-KD (K376R), pHA-Hsp27, and empty control plasmid pcDNA3.1. The transfected cells were heated in water bath at 43°C for 20 min, 40 min and 60 min. Assessments of apoptosis and redistribution of mitochondrial cardiolipin were performed by flow cytometry. PLS3, PKC-δ, Hsp27, phosphorylation of PLS3 and PLS3/PKC-δ interaction were detected by western blotting.Results: In our study the results show that elevated levels of the wild-type PLS3, but not the PLS3 (T21A) mutant, is able to increase hyperthermia-induced CL translocation and apoptosis. Wild-type PKC-δ facilitates PLS3 phosphorylation, PKC-δ/PLS3 interaction, and CL translocation, which consequently promote apoptosis. In contrast, heat shock protein 27 (Hsp27) blocks PKC-δ-induced PLS3 phosphorylation, suppresses PKC-δ/PLS3 interaction and CL translocation, and inhibits apoptosis.Conclusions: Our findings suggest that phosphorylation of PLS3 by PKC-δ is involved in the hyperthermia-induced apoptotic signal transduction pathway in Tca8113 cells, and that Hsp27 blocks this pathway to suppress hyperthermia-induced apoptosis.

Evolving a robust signal transduction pathway from weak cross‐talk

We have evolved a robust two-component signal transduction pathway from a sensor kinase (SK) and non-partner response regulator (RR) that show weak cross-talk in vitro and no detectable cross-talk in vivo in wild-type strains. The SK, CpxA, is bifunctional, with both kinase and phosphatase activities for its partner RR. We show that by combining a small number of mutations in CpxA that individually increase phosphorylation of the non-partner RR OmpR, phosphatase activity against phospho-OmpR emerges. The resulting circuit also becomes responsive to input signal to CpxA. The effects of combining these mutations in CpxA appear to reflect complex intragenic interactions between multiple sites in the protein. However, by analyzing a simple model of two-component signaling, we show that the behavior can be explained by a monotonic change in a single parameter controlling protein–protein interaction strength. The results suggest one possible mode of evolution for two-component systems with bifunctional SKs whereby the remarkable properties and competing reactions that characterize these systems can emerge by combining mutations of the same effect.

Reduced Production of RNA Transcripts from Individual DNA Plasmids Given in a Multivalent DNA Vaccine Formula

We conducted transient transfection studies using two DNA vaccines constructs encodingtwo Plasmodium falciparum surface proteins, PfCSP and PfSSP2, and UM449 melanoma cells todetermine transcription and translation efficiencies. Plasmids were transfected individually or incombination with an empty control plasmid with and without a functional CMV IE promoter.Western blot analysis using NSF1, a monoclonal antibody specific for PfCSP, and UM449 celllysate revealed an abrogation in expression of PfCSP when a plasmid carrying the Pfcsp genewas co-transfected with an empty control plasmid with a functional CMV IE promoter. When acontrol plasmid without a functional CMV IE promoter was substituted in the expression study,normal levels of PfCSP were detected by Western blot. Total RNA was isolated followingtransfection and reverse transcriptase quantitative (RTQ)-PCR was performed. Levels of Pfcspand Pfssp2 transcripts decreased significantly when co-transfected with a control plasmidcontaining a functional CMV IE promoter while transcript levels of Pfcsp and Pfssp2 weresignificantly higher in cells co-transfected with a control plasmid without a functional CMV IEpromoter. The presence of multiple copies of a functional CMV IE promoter leads to a decreasein expression of malaria antigens present in a multivalent vaccine mixture when transfected invitro.

In vivo administration of plasmid DNA encoding recombinant immunotoxin DT390-IP-10 attenuates experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune demyelinating disease. The expression of chemokine receptor CXCR3 on activated T cells is crucial to direct the migration of effector cells into the inflammatory sites and initiate EAE. In this study we tested the effect of a novel recombinant immunotoxin targeting CXCR3 + cells for EAE prevention. The immunotoxin construct DT390-IP-10-SRα consisted of interferon gamma-inducible protein 10 (IP-10), a ligand of CXCR3, as the targeting moiety, and a truncated diphtheria toxin (DT390) as the toxic moiety. In vitro transfection of DT390-IP-10-SRα into NIH3T3 cells resulted in expression of DT390-IP-10 which proved highly toxic to activated T cells. To evaluate the effect of DT390-IP-10-SRα on EAE prevention in vivo, cationic liposome-embedded DT390-IP-10-SRα was injected into the muscle of hind limbs of C57BL/6 mice immunized by myelin basic protein (MBP). DT390-IP-10-SRα-treated mice showed a delayed onset of EAE and milder symptoms compared to the mice treated with empty control plasmid or PBS alone. Immunohistochemical staining detected significantly reduced infiltrating CXCR3 + cells in the inflammatory lesions of CNS from immunotoxin treated mice as compared to the controls. This study suggests that targeting CXCR3 + T cells with recombinant immunotoxin could be achieved in vivo to delay and ameliorate murine EAE.

A role for hSwi/Snf in G2‐M cell cycle arrest and nucleotide excision repair

Mammalian cells exhibit complex cellular responses to genotoxic stress, including cell cycle checkpoints, DNA repair and apoptosis. Inactivation of these important biological events may result in genomic instability and cell transformation, as well as alterations of therapeutic sensitivity. Gadd45a, a p53- and BRCA1-regulated stress-inducible gene, has important roles in nucleotide excision repair (NER), chromatin remodeling, and G2-M cell cycle arrest. The SW-13 cell line, derived from a small-cell carcinoma of the adrenal cortex, lacks a functional SWI/SNF remodeling complex due to the absence of BRG1 and hBrm proteins while they express the remaining Swi/Snf proteins required for complex formation. We have established a stable SW-13 cell line with constitutive Brg1 expression. Significantly, we observed that introduction of BRG1 into SW-13 cells stimulates expression of GADD45. GADD45 is not detectable in SW-13 cells with the empty control plasmid vector. Importantly, SW-13 vector cells are very sensitive to 254 nm UV irradiation, but cells become very resistant to UV when Brg1 is introduced. These findings support a role for hSwi/Snf in NER. Our newly discovered connection between Brg1 and GADD45a also suggests a role for Swi/Snf in G2-M cell cycle checkpoint.

Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1

Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plasmid-containing cells when plasmid copy number decreases, cleaving not only host- but also a specific plasmid-encoded mRNA at the longer and more specific target sequence UUACU. This specific cleavage by Kid inhibits bacterial growth and, at the same time, helps to restore the plasmid copy number. Kid targets a plasmid RNA that encodes a repressor of the synthesis of an R1 replication protein, resulting in increased plasmid DNA replication. This mechanism resembles that employed by some human herpesviruses to regulate viral amplification during infection.

The FK506 Binding Protein Fpr3 Counteracts Protein Phosphatase 1 to Maintain Meiotic Recombination Checkpoint Activity

The meiotic recombination checkpoint delays gamete precursors in G2 until DNA breaks created during recombination are repaired and chromosome structure has been restored. Here, we show that the FK506 binding protein Fpr3 prevents premature adaptation to damage and thus serves to maintain recombination checkpoint activity. Impaired checkpoint function is observed both in cells lacking FPR3 and in cells treated with rapamycin, a small molecule inhibitor that binds to the proline isomerase (PPIase) domain of Fpr3. FPR3 functions in the checkpoint through controlling protein phosphatase 1 (PP1). Fpr3 interacts with PP1 through its PPIase domain, regulates PP1 localization, and counteracts the activity of PP1 in vivo. Our findings define a branch of the recombination checkpoint involved in the adaptation to persistent chromosomal damage and a critical function for FK506 binding proteins during meiosis.

Intrathymic delivery of plasmid-encoding endoplasmic reticulum signal-sequence-deleted MHC class I alloantigen can induce long-term allograft survival

Abstract Intrathymic (IT) delivery of donor alloantigen is a potent strategy to induce operational tolerance. In this study we determined whether this effect was dependent on direct aliorecognition of the tolerogen. Ten microgrammes of plasmid, encoding either the wildtype major histocompatibility complex (MHC) class I molecule Kb or a truncated form in which the signal sequence for translocation into the endoplasmic reticulum was deleted, preventing cell surface expression and direct allorecognition of the tolerogen, was administered intrathymically to CBA.Ca (H2k) recipients. In addition, recipients were treated with anti-CD4 antibody (YTA3.1) at the time of IT injection and underwent transplantation 28 days later with a fully mismatched C57BL/10 (H2b) cardiac allograft. Wildtype, as well as truncated Kb genes, were able to induce long-term survival of the cardiac allografts, in contrast to empty control plasmid. Reversetranscriptase PCR showed expression of the Kb genes for up to 28 days in thymus and spleen of pretreated recipients. These data show that direct allorecognition of the tolerogen was not required for the induction of long-term allograft survival following the introduction of plasmid-encoded MHC alloantigen into the thymus.

Intrathymic delivery of plasmid-encoding endoplasmic reticulum signal-sequence-deleted MHC class I alloantigen can induce long-term allograft survival.

Intrathymic (IT) delivery of donor alloantigen is a potent strategy to induce operational tolerance. In this study we determined whether this effect was dependent on direct allorecognition of the tolerogen. Ten microgrammes of plasmid, encoding either the wildtype major histocompatibility complex (MHC) class I molecule K(b) or a truncated form in which the signal sequence for translocation into the endoplasmic reticulum was deleted, preventing cell surface expression and direct allorecognition of the tolerogen, was administered intrathymically to CBA.Ca (H2(k)) recipients. In addition, recipients were treated with anti-CD4 antibody (YTA3.1) at the time of IT injection and underwent transplantation 28 days later with a fully mismatched C57BL/10 (H2(b)) cardiac allograft. Wildtype, as well as truncated K(b) genes, were able to induce long-term survival of the cardiac allografts, in contrast to empty control plasmid. Reverse-transcriptase PCR showed expression of the K(b) genes for up to 28 days in thymus and spleen of pretreated recipients. These data show that direct allorecognition of the tolerogen was not required for the induction of long-term allograft survival following the introduction of plasmid-encoded MHC alloantigen into the thymus.

Gene transfer of inducible nitric oxide synthase complementary DNA regresses the fibrotic plaque in an animal model of Peyronie's disease.

The goal of the present study was to investigate the antifibrotic role of inducible nitric oxide synthase (iNOS) in Peyronie's disease (PD) by determining whether a plasmid expressing iNOS (piNOS) injected into a PD-like plaque can induce regression of the plaque. A PD-like plaque was induced with fibrin in the penile tunica albuginea of mice and then injected with a luciferase-expressing plasmid (pLuc), either alone or with piNOS, following luciferase expression in vivo by bioluminescence imaging. Rats were treated with either piNOS, an empty control plasmid (pC), or saline. Other groups were treated with pC or piNOS, in the absence of fibrin. Tissue sections were stained for collagen, transforming growth factor (TGF) beta1, and plasminogen-activator inhibitor (PAI-1) as profibrotic factors; copper-zinc superoxide dismutase (CuZn SOD) as scavenger of reactive oxygen species (ROS); and nitrotyrosine to detect nitric oxide reaction with ROS. Quantitative image analysis was applied. Both iNOS and xanthine oxido-reductase (XOR; oxidative stress) were estimated by Western blot analysis. Luciferase reporter expression was restricted to the penis, peaked at 3 days after injection, but continued for at least 3 wk. In rats receiving piNOS, iNOS expression also peaked at 3 days, but expression decreased at the end of treatment, when a considerable reduction of plaque size occurred. Protein nitrotyrosine, XOR, and CuZn SOD increased, and TGFbeta1 and PAI-1 decreased. The piNOS gene transfer regressed the PD plaque and expression of profibrotic factors, supporting the view that endogenous iNOS induction in PD is defense mechanism by the tissue against fibrosis.

A Novel Zinc Finger Protein Interacts with Receptor-interacting Protein (RIP) and Inhibits Tumor Necrosis Factor (TNF)- and IL1-induced NF-κB Activation

Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-kappa B activation. In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified ZIN (zinc finger protein inhibiting NF-kappa B), a novel protein that specifically interacts with RIP. ZIN contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus. Overexpression of ZIN inhibits RIP-, IKK beta-, TNF-, and IL1-induced NF-kappa B activation in a dose-dependent manner in 293 cells. Domain mapping experiments indicate that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-kappa B activation. Overexpression of ZIN also potentiates RIP- and TNF-induced apoptosis. Moreover, immunofluorescent staining indicates that ZIN is a cytoplasmic protein and that it colocalizes with RIP. Our findings suggest that ZIN is an inhibitor of TNF- and IL1-induced NF-kappa B activation pathways.