Minimal piggyBac vectors for chromatin integration.

We describe novel transposon piggyBac vectors engineered to deliver transgenes as efficiently as currently available piggyBac systems, but with significantly less helper DNA co-delivered into the host genome. To generate these plasmids, we identified a previously unreported aspect of transposon biology, that the full-length terminal domains required for successful plasmid-to-chromatin transgene delivery can be removed from the transgene delivery cassette to other parts of the plasmid without significantly impairing transposition efficiency. This is achieved by including in the same plasmid, an additional helper piggyBac sequence that contains both long terminal domains, but is modified to prevent its transposition into the host genome. This design decreases the size of the required terminal domains within the delivered gene cassette of the piggyBac vector from about 1500 to just 98 base pairs. By removing these sequences from the delivered gene cassette, they are no longer incorporated into the host genome which may reduce the risk of target cell transformation.

Mifepristone increases gamma-retroviral infection efficiency by enhancing the integration of virus into the genome of infected cells.

Gamma-retroviruses are commonly used to deliver genes to cells. Previously, we demonstrated that the synthetic anti-glucocorticoid and anti-progestin agent, mifepristone, increased gamma-retroviral infection efficiency in different target cells, independent of viral titer. In this study, we examine how this occurs. We studied the effect of mifepristone on different steps of viral infection (viral entry, viral survival, viral DNA synthesis and retrovirus integration into the host genome) in three distinct retroviral backbones using different virus recognition receptors. We also tested the potential role of glucocorticoid and progesterone receptors in mediating mifepristone's ability to increase gamma-retroviral infectivity. We show that mifepristone increases gamma-retroviral infection efficiency by facilitating viral integration into the host genome and that this effect seems to be due to mifepristone's anti-glucocorticoid, but not its anti-progestin, activity. These results suggest that inhibition of the glucocorticoid receptor enhances retroviral integration into the host genome and indicates that cells may have a natural protection again retroviral infection that may be reduced by glucocorticoid receptor antagonists.

Primary neural stem/progenitor cells expressing endostatin or cytochrome P450 for gene therapy of glioblastoma.

Despite recent technical improvements in surgical excision techniques and adjuvant radio- and chemotherapy, the clinical outcome of patients with grade IV astrocytoma (glioblastoma) remains very poor, with a median survival of less than 12 months. A promising approach to therapy employs gene-engineered neural stem/progenitor cells (NSCs) as a cellular therapeutic delivery system, to track glioblastoma cells and deliver anticancer molecules. However, most results on their tumor tropism have been derived by immortalized NSCs. We now report that primary murine gene-engineered NSCs displayed in vivo tropism for glioblastoma cells. Ten days after injection into the brain, many NSCs continued to express the transgene and the NSC marker, nestin. NSCs transduced with a retroviral vector co-expressing a secretable form of human endostatin and eGFP (NSC/endo-eGFP) released potentially antiangiogenetic concentrations of endostatin into the culture medium. Conditioned medium of NSC/endo-eGFP cells inhibited the growth of mouse pulmonary microvascular endothelial cells (PMVECs). A good correlation between endostatin levels and PMVEC growth inhibition was observed. In NSCs co-expressing cytochrome P450 2B6 (CYP2B6) and eGFP (NSC/CYP2B6-eGFP), the forced expression of CYP2B6 resulted in intracellular activation of CPA and subsequent cell death. In the presence of NSC/CYP2B6-eGFP, we observed CPA cytotoxicity to DsRed-expressing U87Mg glioma cells. In vivo treatment of intracranial GL-261 glioblastoma with NSC/endo-eGFP caused a 65% reduction in tumor size, compared to untreated control mice or mice treated with NSC/eGFP cells. Our data suggest that primary NSCs transduced with retroviral vectors expressing endostatin and/or CYP2B6 have a potential role in glioblastoma therapy.

[The role of Cd-binding proteins and phytochelatins in the formation of cadmium resistance in Nicotiana plumbaginifolia cell lines].

Nicotiana plumbaginifolia callus lines with the equal resistance to cadmium have been produced under different selective conditions--either without inhibition of the phytochelatin synthesis (line Cd-R) or in the presence of the inhibitor butionine sulfoximine (line Cd-Ri). The level of phytochelatin synthesis in the line Cd-R five-fold exceeded the control value and in the line Cd-Ri it was twice as much as in the control. It was shown that in the control line mainly three cadmium-binding proteins are expressed of the molecular weihgts 41, 34 and 19 kD. The common feature of the both resistant lines is the expression of the cadmium-binding proteins of 40, 37 and 19 kD. The resistant lines differ with respect to the synthesis of relatively low-molecular cadmium-binding proteins. The proteins of the molecular weights 12.5, 11.5 and 9 kD are expressed in the line Cd-R, while the proteins of 13 and 10 kD are expressed in the line Cd-Ri. It was supposed that both the phytochelatins and the Cd-binding proteins contribute to the resisitance of N. plumbaginifolia callus lines to cadmium and the lack of the phytochelatins can be equilibrated by the changes in the low-molecular Cd-binding protein synthesis.

Dexamethasone can stimulate G1-S phase transition in human airway fibroblasts in asthma.

Corticosteroids are the first line of therapy for asthma. Whether they alter the progression of airway remodelling in asthma is, as yet, unknown. To determine whether corticosteroids could alter the fibroblast cell cycle the current authors studied the effect of dexamethasone on cultured airway fibroblasts obtained from nine mild-to-moderate, steroid-naïve asthmatics (forced expiratory volume in one second 78+/-4% predicted), and seven normal controls. Fibroblasts were cultured from endobronchial biopsies obtained via bronchoscopy. Cells were exposed to dexamethasone (10(-9)-10(-7) M) and studied at 72 h to determine differences in progression through the cell cycle. In asthmatic fibroblasts, dexamethasone, at concentrations of 10(-8)M and 10(-7)M, nearly doubled the number of cells in the S phase (17.8+/-3.0% and 18.4+/-3.1%, respectively) compared with untreated fibroblasts (10.3+/-1.4%). There was no significant effect in normal control fibroblasts. Dexamethasone induced hyperphosphorylation of the tumour suppressor, retinoblastoma (RB) in asthmatic fibroblasts; fibroblasts from normal controls had significantly less hyperphosphorylation of RB. No difference in protein expression of the CCAAT/enhancer binding protein alpha between the two groups was detected. This study suggests that dexamethasone can stimulate G1-S phase cell cycle transition in human airway fibroblasts obtained from asthmatics. Whether this leads to enhanced airway remodelling in some individuals remains to be determined.

Cardiotrophin-1 in choroid plexus and the cerebrospinal fluid circulatory system.

There is a growing recognition of choroid plexus functioning as a source of neuropeptides, cytokines and growth factors in cerebrospinal fluid (CSF) with diffusional access into brain parenchyma. In this study, choroid plexus and other components of the CSF circulatory system were investigated by Western blotting, reverse transcriptase polymerase chain reaction and immunohistochemistry for production of interleukin-6-related cytokines characterized by neuroactivity [cardiotrophin-1 (CT-1), ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M] and signaling through the gp130/leukemia inhibitory factor receptor-beta receptor heterodimer. Western blot analysis showed that CT-1 was the only cytokine family member detectable in adult rat choroid plexus, as in leptomeninges. The specificity of detection was verified with blots of the same tissues from CT-1-deficient mice. Levels of both CT-1 mRNA and protein were constitutively high in rat from birth through adulthood in choroid plexus, up-regulated postnatally in leptomeninges and undetectable in brain parenchyma. Using antigen retrieval, CT-1 immunolocalized to choroid epithelial cells in all choroid plexuses in addition to leptomeninges (arachnoid and pial-glial membranes). Ependymal cells lining the ventricular neuroaxis, unlike the central canal, were also CT-1-immunoreactive. Western blots indicated rat choroid epithelial cells express and release CT-1 immunoreactivity under defined culture conditions and also revealed the presence of a CT-1-like protein in human choroid plexus and CSF. Previously, CT-1 has been conceptualized to function as a target-derived factor for PNS neurons. Our study clearly demonstrates production of CT-1 in the postnatal and adult CNS, specifically by cell types comprising the blood-CSF barrier, and its accumulation in ventricular ependyma. This finding has broad implications for CT-1 functioning apart from other leukemia inhibitory factor receptor ligands as a CSF-borne signal of brain homeostasis, one possibly involving regulation of the barrier itself, the ependyma or target cells in the surrounding parenchyma, including the subventricular zone. A rationale for studies examining CT-1-deficient mice in these respects is provided by the data.

Effect of ischemia, calcium depletion and repletion, acidosis and hypoxia on cellular taurine content.

Occlusion of the left main coronary artery led to a time-dependent release of taurine from the heart. Upon reperfusion, there was a second phase of taurine release, which exceeded the amount of taurine that exited the heart during the 45 min ischemic insult. To obtain information on the mechanism underlying the release of taurine, three variables were examined, acidosis, hypoxia and calcium overload. It was found that large amounts of taurine also leave the cell during the calcium paradox, a condition induced by perfusing the heart with calcium containing buffer following a period of calcium free perfusion. However, little taurine effluxes the hearts exposed to buffer whose pH was lowered to 6.6. Isolated neonatal cardiomyocytes subjected to chemical hypoxia also lost large amounts of taurine. However, the amount of taurine leaving the cells appeared to be correlated with the intracellular sodium concentration, [Na(+)](i). The data suggest that taurine efflux is regulated by [Na(+)](i) and cellular osmolality, but not by cellular pH.

4-Hydroxynonenal, a lipid peroxidation byproduct of spinal cord injury, is cytotoxic for oligodendrocyte progenitors and inhibits their responsiveness to PDGF.

Oligodendroglial reactions to compression injury of spinal cord include apoptosis, secondary demyelination, and remyelination failure. Within hours after contusion, the membrane lipid peroxidation (MLP) byproduct, 4-hydroxynonenal (HNE), increases rapidly in gray matter and thereafter in white matter tracts beyond the initial lesion level. Considering that HNE is a mediator and marker of neuronal MLP toxicity in various neurodegenerative conditions, the present study examined its effect on the regeneration potential of oligodendrocyte progenitors, as defined by their capacity to survive, proliferate and migrate in primary culture. Treatment of oligodendroblasts with HNE evoked a time- and dose-dependent cytotoxicity resembling apoptosis at aldehyde concentrations known to be produced by neurons and achieved in tissue undergoing peroxidative injury. In addition, sublethal concentrations of HNE inhibited the mitogenic and chemotactic responses of more immature progenitors to platelet-derived growth factor. These effects appear to be mediated in part by the formation of HNE adducts with progenitor proteins located within the plasma membrane and cytoplasmic compartments. Our data are the first to show that HNE can have direct, deleterious effects on oligodendrocyte precursors. The present study also suggests a mechanism by which the striking accumulation of HNE in white matter tracts surrounding the site of spinal cord compression injury and in other ischemic-hypoxic insults associated with MLP could suppress the potential regenerative response of endogenous oligodendrocyte progenitor cells.

Cardioprotective effect of chronic hyperglycemia: effect on hypoxia-induced apoptosis and necrosis.

It is generally accepted that mild forms of diabetes render the heart resistant to an ischemic insult. Because myocytes incubated chronically in medium containing high concentrations of glucose (25 mM) develop into a diabetes-like phenotype, we tested the hypothesis that high-glucose treatment diminishes hypoxia-induced injury. In support of this hypothesis, we found that cardiomyocytes incubated for 3 days with medium containing 25 mM glucose showed less hypoxia-induced apoptosis and necrosis than cells exposed to medium containing 5 mM glucose (control). Indeed, whereas 27% of control cells became necrotic after 1 h of chemical hypoxia with 10 mM deoxyglucose and 5 mM amobarbital (Amytal), only 11% of the glucose-treated cells became necrotic. Similarly, glucose treatment reduced the extent of apoptosis from 32% to 12%. This beneficial effect of glucose treatment was associated with a 40% reduction in the Ca(2+) content of the hypoxic cell. Glucose treatment also mediated an upregulation of the cardioprotective factor Bcl-2 but did not affect the cellular content of the proapoptotic factors Bax and Bad. Nonetheless, the phosphorylation state of Bad was shifted in favor of its inactive, phosphorylated form after high-glucose treatment. These data suggest that glucose treatment renders the cardiomyocyte resistant to hypoxia-induced apoptosis and necrosis by preventing the accumulation of Ca(2+) during hypoxia, promoting the upregulation of Bcl-2, and enhancing the inactivation of Bad.

[Inhibition of lipoxygenase activity reduces radiation-induced DNA fragmentation in lymphocytes]. / Ingibirovanie lipoksigenaznoi aktivnosti snizhaet indutsirovannuiu radiatsiei fragmentatsiiu DNK limfotsitov.

Lypoxygenase activity of lymphocytes towards exogenous substrate linolic acid and the level of endogenous arachidonate metabolite 15-HETE are increased within 6 h and decreased below the control level at 24 h after 1 Gy X-ray irradiation of Wistar rats. DNA fragmentation is stimulated both in vivo within 12 h and during in vitro incubation of lymphocytes, obtained 3 h following irradiation. DNA analysis by agarose gel shows that DNA fragments are multiples of the 200 base pair unit. When 20 microM nordihydroguayaretic acid, a selective inhibitor of arachidonate lypooxidation, was added to the incubation medium, DNA fragmentation was observed to be significantly less, especially at the early steps of incubation.

[Lipoxygenase activity and 15-hete content of rat spleen lymphocytes after exposure to ionizing radiation]. / Lipoksigenaznaia aktivnost' i soderzhanie 15-hete v limfotsitakh selezenki krys pri deistvii ioniziruiushchei radiatsii.

Pretreatment of rat spleen lymphocytes with 0.5 mM A23187 had no influence on the prostaglandins E2 and F2 alpha content, but was followed by the increasing of both 15-HETE and leukotriene B4 levels. Lypoxygenase activity of lymphocytes towards exogenous substrate linoleic acid was increased within 3-12 h after 1 Gy irradiation and decreased below the control level at 24 h. The changes of lypoxygenase activity correlate with that of 15-HETE content. Additional incubation of the cells, obtained at 3 h after irradiation, was followed by the intensification of the chromatin internucleosomal fragmentation and low-molecular DNA fragments accumulation. When 20 mM nordihydroguaiaretic acid, a selective inhibitor of arachidonate lipooxidation, was added to the incubation medium, DNA fragmentation was observed to be significantly less, especially at the early steps of incubation. These results suggest, that metabolites like H(P)ETE are early endogenous mediators of radiation-induced spleen lymphocytes apoptosis.

[Endonucleolysis of chromatin in rat thymus and spleen lymphocytes after irradiation]. / Endonukleoliz khromatynu u limfotsytakh selezinky ta tymusa shchuriv pislia oprominennia.

The level of soluble DNA fragments (polydesoxynucleotides) in thymus and spleen lymphocytes is enhanced 12 h after the whole-body X-ray irradiation in dose of 0.5 and 1 Gy. The level of Ca2+, Mg(2+)-DNAase activity in lysed cells extracts, measured with calf spleen DNA, was enhanced in the region of 0.1-0.5 mM CaCl2 in incubation medium and decreased at 5 mM CaCl2. Monitoring of DNA fragmentation by electrophoresis in agarose gels showed the presence of fragments with approximately 200, 400, 800 bp length. X-ray induced DNA fragmentation is supposed to be associated with Ca2+, Mg(2+)-endonuclease activation.