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PubMed2006

Adenoviral vector platform for transduction of constitutive and regulated tricistronic or triple-transcript transgene expression in mammalian cells and microtissues.

BACKGROUND: Adenoviral particles can efficiently transduce a broad spectrum of cell types, so they are widely used in basic research and clinical trials. METHODS: We have developed a novel adenoviral vector platform for delivery of constitutive or streptogramin-inducible expression of up to three therapeutic transgenes into a variety of murine and human cell lines, primary cells and microtissues. RESULTS: Coordinated expression of three independent transgenes in a compact genetic format was achieved by two different expression configurations: (i) The multicistronic expression format consisting of a single constitutive (simian virus 40 promoter, P(SV40); murine or human cytomegalovirus immediate-early promoter, P(mCMV), P(hCMV)) or regulated (streptogramin-inducible) promoters (P(PIR)ON2) driving the expression of a single multicistronic transcript of which the first cistron is translated in a cap-dependent manner and the two subsequent ones by internal ribosome entry site (IRES)-mediated translation initiation. (ii) The triple-transcript expression configuration, in which a combination of well-established (P(SV40), P(hCMV), P(mCMV)) and novel synthetic constitutive promoters (P(GTX)) control transcription of three expression units. The constitutive multigene expression design enabled coordinated high-level expression of the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY), the human vascular endothelial growth factor 121 (VEGF(121)) and the human placental secreted alkaline phosphatase (SEAP) in monolayer populations and microtissues of Chinese hamster ovary cells (CHO-K1), human fibrosarcoma cells (HT-1080), primary neonatal rat cardiomyocytes (NRCs) and primary human aortic fibroblasts (HAFs). Streptogramin-inducible tricistronic SAMY-VEGF(121)-SEAP expression provided excellent regulation performance-high-level induction in the presence of the streptogramin antibiotic pristinamycin I (PI), near-undetectable basal expression in the absence of PI, optimal adjustability and perfect reversibility-in all cell types, in particular in NRCs and NRC-derived myocardial microtissues. CONCLUSIONS: Triple-transcript and tricistronic expression configurations conserve the DNA packaging capacity of the size-constrained viral transduction systems and enable coordinated and regulated expression of up to three therapeutic transgenes for concerted clinical interventions in future gene therapy scenarios.

Adenoviridae↗
PubMed2006

Region-specific constitutive gene expression in the adult porcine meniscus.

The knee meniscus exhibits extensive spatial variations in native healing capacity, biochemical composition, and cell morphology that suggest the existence of distinct phenotypes for meniscus cells. Constitutive gene expression levels of appropriate extracellular matrix proteins may serve as useful molecular markers of cellular phenotypes; however, relatively little is known of variations in the gene expression for meniscus cells of different regions of the tissue. The objective of the present study was to evaluate constitutive differences between radial inner and outer regions in gene expression for extracellular matrix proteins relevant to the meniscus. A secondary objective was to determine if these region-specific differences in gene expression are maintained after periods of monolayer culture. The innermost regions of the meniscus were found to constitutively express higher mRNA levels for proteins highly expressed in articular cartilage, including aggrecan, type II collagen, and NOS2. In contrast, the outer meniscus was found to contain higher gene expression for proteins associated with fibrous tissues including type I collagen, and the proteases MMP2 and MMP3. Isolated inner and outer meniscus cells maintained these region-specific gene expression patterns for collagens and proteoglycans during short-term monolayer culture. The results provide new information that suggests the utility of constitutive gene expression levels as molecular markers to distinguish tissue and cells of the inner and outer meniscus.

Animals↗
PubMed1994

Constitutive AP-1 DNA binding and transactivating ability of malignant but not benign mouse epidermal cells.

The mouse epidermal cell line 308 contains an activated Ha-ras gene and forms benign papillomas when transplanted to the skin of athymic nude mice. A radiation-associated malignant variant of this cell line, 308-10Gy5, has been isolated and shown to form squamous cell carcinomas in nude mice. To further examine the molecular events involved in malignant conversion of 308-10Gy5, we assessed the activator protein-1 (AP-1) binding and transactivating ability of 308 and 308-10Gy5. In nuclear protein extracts of 308, AP-1 sequence-specific binding to an oligonucleotide containing a single high-affinity AP-1 binding site was induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, as determined by gel shift analysis. Nuclear extracts of 308-10Gy5 bound to the AP-1 oligonucleotide without treatment with tumor promoters. Not only was sequence-specific AP-1 DNA binding constitutively active in malignant versus benign tumor cells, but so was transactivation of a unique AP-1-responsive chloramphenicol acetyltransferase reporter construct, pTiCTaK. Constitutive transactivation of this AP-1-responsive reporter construct was observed in the malignant but not the benign tumor cells. Furthermore, steady-state transcript levels of the tumor-associated AP-1-responsive genes stromelysin, urokinase-type plasminogen activator, c-jun, and c-fos were higher in malignant 308-10Gy5 cells than in benign 308 cells. These results suggest that acquisition of constitutive AP-1 DNA binding and transactivation can result in sustained deregulation of gene expression. While malignant progression in keratinocytes is probably not due solely to the acquisition of constitutive cellular AP-1 activity, the effect of deregulated expression of AP-1-regulated genes, especially basement membrane-degrading enzymes, may be functionally related to malignant conversion.

Animals↗
PubMed2005

EGFR and Her-2 regulate the constitutive activation of NF-kappaB in PC-3 prostate cancer cells.

BACKGROUND: The mechanism through which NF-kappaB (NF-kappaB) is constitutively activated in prostate cancer cells remains unclear. We investigated whether members of the ErbB family of epidermal growth factor receptors (EGFR) are involved in the constitutive activation of NF-kappaB in prostate cancer cell lines. METHODS AND RESULTS: EGFR, Her-2, and ErbB3 are expressed and constitutively activated in PC-3, DU145, and LNCaP prostate cancer cells lines. Using several pharmacological ErbB inhibitors, we demonstrate that EGFR and Her-2 are involved in the constitutive activation of NF-kappaB in PC-3 cells through two different mechanisms. EGFR activates NF-kappaB through the phosphorylation of IkappaBalpha on serines 32/36 thereby influencing the nuclear translocation of the p65 subunit. In contrast, Her-2 activates NF-kappaB independently of IkappaBalpha phosphorylation on serines 32/36. CONCLUSION: This study directly implicates ErbB receptors in the activation of NF-kappaB in PC-3 prostate cancer cells.

ErbB Receptors↗
PubMed1994

Differential regulation of constitutive and inducible nitric oxide production by inflammatory stimuli in murine endothelial cells.

The murine vascular endothelial cell line send1 expresses both constitutive and inducible nitric oxide (NO) synthases. Interferon-gamma (IFN gamma) or endotoxin (LPS) alone inhibited constitutive NO production in a dose and time dependent manner. Addition of L-arginine had no influence on the decrease of NO production caused by IFN gamma or LPS. On the other hand, IFN gamma and LPS synergized in the induction of high output NO production. Successive incubations with IFN gamma and LPS in different sequences revealed IFN gamma as the time setting signal for the induction of NO synthesis. These results demonstrate that LPS and IFN gamma have a direct suppressive effect on constitutive NO synthase while at the same time they strongly activate inducible NO production. Thus inflammatory stimuli trigger murine vascular endothelial cells to switch from constitutive to inducible NO synthase activity.

Animals↗
PubMed1995

Hierarchal constitutive phosphorylation of the vesicular stomatitis virus P protein and lack of effect on P1 to P2 conversion.

In vitro reconstitution of a transcriptionally active VSV polymerase complex (P:L) reportedly requires phosphorylation of the N-terminal domain of P by CKII. Two constitutively phosphorylated sites have been implicated in this activation for both VSV Indiana and New Jersey serotype P proteins. We show here that, in contrast to New Jersey, the Indiana P protein is constitutively phosphorylated on three sites in vivo. The evidence rests on assessing the phosphorylation status of transfected P gene constructs containing all possible combinations of Ala substitutions at Ser60, Thr62, and Ser64. All mutants containing the T62A substitution showed a reduced level of phosphorylation and yielded no P-Thr. Surprisingly the S60A/S64A mutant behaved like the triple substitution and displayed no significant phosphorylation, while the S64A mutant yielded no P-Thr. Phosphorylation of Thr62 therefore depended on prior modification of Ser64. We also tested the ability of our mutant P proteins to convert to the more highly phosphorylated P2 species, a modification essential for transcription in the New Jersey serotype and thought to be carried out by an L-protein-associated kinase. All of our transfected mutant P proteins readily converted to P2 in the presence or absence of L cotransfection, and the latter had no significant effect on P phosphorylation. We conclude that VSV Indiana P protein differs in significant ways from New Jersey P. It is hierarchically and constitutively phosphorylated on a cluster of three sites, not two, suggesting that an additional kinase may be involved. Moreover, Indiana P1 to P2 conversion is independent of prior constitutive phosphorylation and does not require the presence of L protein.

Alanine↗
PubMed1995

Agonist induced constitutive receptor activation as a novel regulatory mechanism. Mu receptor regulation.

We propose the hypothesis that certain G protein coupled receptors can become constitutively activated during agonist stimulation so that the receptor remains active even after the agonist is removed. This new paradigm of receptor regulation may account for some long term effects of neurotransmitters and hormones. We have tested the hypothesis that constitutive mu receptor activation represents a crucial step driving narcotic tolerance and dependence. Our results indeed support the conversion of mu to a constitutively active state, mu*, observed in neuroblastoma SK-N-SH and SH-SY5Y tissue culture, in U293 cells transfected with the mu receptor gene, and in vivo. Constitutive mu activation may result from receptor phosphorylation to yield mu*, and further, in vivo studies indicate that formation of mu* could account for narcotic tolerance and dependence.

Animals↗
PubMed1980

Isolation of mutants of Escherichia coli with increased resistance to alkylating agents: mutants deficient in thiols and mutants constitutive for the adaptive response.

A search has been made for mutants of E. coli that are constitutive for the adaptive response to alkylating agents. When selection was for resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) the resulting strains all proved to be low in intracellular thiols. As MNNG has to be converted by thiols to the proximal mutagen methylnitrosamine, these strains were resistant simply because their low thiol levels made them less readily methylated by MNNG. The selection was therefore undertaken for strains resistant to N-methylnitrosourea (MNU) because this alkylating agent does not require activation by thiols. Four MNU resistant mutants proved to be constitutive for the adaptive response. Following exposure to (3H) MNNG, they showed the same low ratio of O6-methylguanine (O6MeG) to 7-methylguanine (7MeG) as adapted wild type bacteria. The ratio of 3-methyladenine (3MeA) to 7MeG was the same in the wild type, the adapted wild type, and the constitutive strains. When exposed to very high MNNG doses, the constitutive mutants were even more resistant to mutation and killing than the adapted wild type. They have an even greater ability than the adapted wild type to remove O6MeG from methylated DNA (Lindahl personal communication).

Alkylating Agents↗
PubMed1976

Constitutional chromosomal breakage.

There were 18 individuals found to have a constitutional chromosome fragility causing an increase in break frequency. For each chromosome the breakpoint is always the same, whether it involves chromosomes from the same person, the same family, or different families. The fragile points are bands 10q24, 12q13, 16q21, 17p12, and Xq27. Autosomal constitutional fragility does not seem to have a phenotypic correspondence. They were found mostly in parents of children with chromosomal abnormalities or in couples with a history of repeated spontaneous abortions which permits one to raise the possibility of an interchromosomal effect. The six constitutional chromosomal fragilities of the X chromosome had in common the association of mental deficiency, delayed speech, and large malformed ears. The break points in constitutional chromosomal fragility were compared to those of spontaneous breaks in vitro, to those induced by X-rays, and to those in Fanconi's anemia. The theoretical consequences of these structural abnormalities are discussed as well as what to do about them when they are found.

Abortion, Habitual↗
PubMed1985

DNase I sensitivity in facultative and constitutive heterochromatin.

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes in Microtus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. In Microtus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.

Animals↗
PubMed1995

Inhibition of constitutive and inducible cyclooxygenase activity in human platelets and mononuclear cells by NSAIDs and Cox 2 inhibitors.

A range of NSAIDs and reported Cox 2 selective compounds were tested in human freshly isolated platelets and LPS-stimulated mononuclear cells to determine their potency and selectivity as inhibitors of constitutive (presumably Cox 1) and inducible (presumably Cox 2) cyclooxygenase respectively. All compounds tested were either equipotent at inhibiting constitutive and inducible cyclooxygenase or were selective for the inducible form. The most selective compound was Dup697 and the least selective, ketoprofen. Several compounds only produced a partial inhibition of constitutive cyclooxygenase as the maximum inhibitor concentration achievable in the assay was limited to 1 mM. With the exception of paracetamol, all compounds were able to produce full inhibition curves against the inducible form. Potency estimates against constitutive Cox compare closely with published data but most compounds were consistently more potent against the inducible isoform than in published data for human cloned, microsomal Cox 2. These data suggest that human mononuclear cells are either exquisitely sensitive to some NSAIDs or they may contain another Cox isoform as yet indistinguishable from Cox 2.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2001

Constitutive nitric oxide synthase is associated with retinal vascular permeability in early diabetic rats.

AIMS/HYPOTHESIS: We investigated the association between vascular permeability and constitutive nitric oxide synthase in rats with diabetes for a short duration (2 weeks). METHODS: Retinal vascular permeability was evaluated in rats with diabetes induced by streptozotocin using vitreous fluorophotometry and a small animal adapter. We carried out in situ hybridization and semi-quantitative reverse transcription-polymerase chain reaction to study the expression of endogenous constitutive nitric oxide synthase mRNA in diabetic retinas. We also examined changes in the protein expression of constitutive nitric oxide synthase in diabetic retinas using immunohistochemistry and Western blotting. RESULTS: Retinal vascular permeability was significantly higher in diabetic rats (median, 1.09 arbitrary unit) compared with control rats (median, 0.69 arbitrary unit) (p < 0.05). The expression of both neuronal nitric oxide synthase (NOS) and endothelial nitric oxide synthase mRNA was higher in diabetic retinas than in the retinas of control rats as determined by in situ hybridization and reverse transcription-polymerase chain reaction. Immunohistochemistry and Western blotting also showed that neuronal nitric oxide synthase increased in diabetic retinas. The immunohistochemistry of endothelial nitric oxide synthase indicated that non-vessel tissues increased in diabetic retinas while retinal vessels weakened. Western blotting showed that the amount of endothelial nitric oxide synthase increased. CONCLUSION/INTERPRETATION: These results suggest that increases in both constitutive NOSs (nNOS and eNOS) could be associated with retinal vascular permeability and that NOS is associated with clinical vascular dysfunction in the early stages of diabetes.

Animals↗
PubMed2004

Stable expression of constitutively activated mutant h5HT6 and h5HT7 serotonin receptors: inverse agonist activity of antipsychotic drugs.

RATIONALE: In order to determine the possible relationship between antipsychotic drug properties and inverse agonist activity at h5HT6 and h5HT7 receptors, constitutively activated forms of these receptors were created by site-specific mutagenesis. Typical and atypical antipsychotic drugs were assayed for their potencies as inverse agonists at these mutated receptors. OBJECTIVES: Stable cell lines expressing constitutively activated forms of the h5HT6 and h5HT7 receptors were created. Typical and atypical antipsychotic drugs demonstrating high to moderate affinities for the h5HT6 and h5HT7 receptors were assayed for their potencies in reversing the agonist-independent activity (inverse agonist activity). RESULTS: The E322R h5HT6 mutant and the S267K h5HT7 mutant displayed sufficiently robust agonist-independent activity when expressed in stable cell lines to allow the detailed, concentration-dependent, investigation of the inverse agonist activity of typical and atypical antipsychotic drugs. All the drugs tested displayed inverse agonist activity at both the activated h5HT6 and h5HT7 receptors. Native forms of these receptors did not display any constitutive activity. Interestingly, atypical antipsychotic drugs displayed potent inverse agonist activity, relative to typical antipsychotic drugs, at the h5HT7 receptor. LSD displayed neutral antagonist properties at the mutant h5HT7 receptor. CONCLUSIONS: Site-specific mutations in the third intracellular loop of the G(s)-coupled h5HT6 and h5HT7 receptors produce constitutive activation. Antipsychotic drugs display inverse agonist activity at the activated receptors. The inverse agonist mechanism-of-action of the atypical antipsychotic drugs at the h5HT7 receptors may be different from the typical antipsychotic drugs as these drugs displayed far higher potencies as inverse agonists at the h5HT7 receptor.

Amino Acid Substitution↗
PubMed2005

Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells.

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH-GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20 degrees C for 2 h and then released by warming up to 37 degrees C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH-GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.

Base Sequence↗
PubMed2005

Evolution versus constitution: differences in chromosomal inversion.

We compared the chromosomal breakpoints of evolutionary conserved and constitutional inversions. Multicolor banding and human-specific bacterial artificial chromosomes were applied to map the breakpoints of constitutional pericentric inversions on human chromosomes 2 and 9. For the first time, we present a high-resolution analysis of the breakpoint regions, which are characterized by gene destitution, co-localization with fragile sites, multitude repeats as well as pseudogenes and, remarkably, a large sequence homology to the opposite breakpoint. In contrast, evolutionary inversion breakpoints lack such extensive cross-hybridizing regions and are often associated with fragile sites of the genome and low-copy repeats. These molecular characteristics gave evidence for different types of inversion formation and indicate that evolutionary inversions cannot originate from constitutional inversions like those of chromosomes 2 and 9. Finally, the constitutional inversion breakpoints were investigated on three different great ape species and on four test persons each bearing the same cytogenetically determined inversion on chromosomes 2 and 9, respectively. Our data indicate the existence of different molecular breakpoints for the two variant chromosomes.

Animals↗
PubMed2005

Differential appearance of dynamin in constitutive and regulated exo-endocytosis: a single-cell multiplex RT-PCR study.

Neurons in the central nervous system establish, via their axons and dendrites, an extended network that allows synaptic transmission. During developmental maturation and process outgrowth, membrane turnover is necessary for the enlargement and subsequent growth of axons and dendrites from the perikarya to the target cell (constitutive exocytosis/endocytosis). After targeting and synapse formation, small synaptic vesicles are needed for the quantal release of neurotransmitters from the presynaptic terminal with subsequent recycling by regulated exocytosis/endocytosis. An investigation of the onset of the appearance of mRNA and protein in dissociated cultures of neurons from mouse hippocampus or from chick retina has shown an early abundance of proteins involved in exocytosis, such as syntaxin 1, SNAP-25, and synaptotagmin 1, whereas dynamin 1, a protein necessary for clathrin-mediated endocytosis, can be detected only after neurons have established contacts with neighboring cells. The results reveal that constitutive membrane incorporation and regulated synaptic transmitter release is mediated by the same neuronal proteins. Moreover, the data exclude that dynamin 1 takes part in constitutive recycling before synapse formation, but dynamin 2 is present at this stage. Thus, dynamin 2 may be the constitutive counterpart of dynamin 1 in growing neurons. Synapse establishment is linked to an upregulation of dynamin 1 and thereby represents the beginning of the regulated recycling of membranes back into the presynaptic terminal.

Animals↗
PubMed2006

Constitutive expression of human angiostatin in Pichia pastoris by high-density cell culture.

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 60 h at 30 degrees C. Dissolved oxygen level was maintained at 25-30% and pH was controlled at 5 by the addition of 7 M NH4OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins.

Angiogenesis Inhibitors↗
PubMed2005

Expression of constitutive and inducible chemical defenses in native and invasive populations of Alliaria petiolata.

The Evolution of Increased Competitive Ability (EICA) hypothesis posits that invasive plants in introduced habitats with reduced herbivore pressure will evolve reduced levels of costly resistance traits. In light of this hypothesis, we examined the constitutive and inducible expression of five chemical defense traits in Alliaria petiolata from four invasive North American and seven native European populations. When grown under common conditions, significant variation among populations within continents was found for trypsin inhibitors and peroxidase activity, and glucosinolates and trypsin inhibitors were significantly jasmonate-inducible across populations. Across populations, constitutive levels of glucosinolates and trypsin inhibitors were negatively correlated with their degree of induction, with three North American populations tending to have lower constitutive levels and higher inducibility of glucosinolates than the seven European populations. Alliarinoside and isovitexin 6"-O-beta-glucopyranoside levels were both higher in North American plants than in European plants, but levels of these compounds were generally increased by jasmonate in European plants and decreased by the same treatment in North American plants. Aside from the tendency for invasive populations to have reduced constitutive glucosinolate levels coupled with increased inducibility, little support for the predictions of EICA was evident in the chemical defenses that we studied.

Adaptation, Physiological↗