Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen-limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone-inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb-expressing clone and the parental CHO-tPA expressing cells. The VHb-expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO-tPA culture. (c) 1994 John Wiley & Sons, Inc. 相似文献
New data are provided to show that (i) rat Sertoli cells produce two types of plasminogen activators, tissue type (tPA) and urokinase type (uPA), and a plasminogen activator inhibitor type-1 (PAI-1); (ii) both tPA (but not uPA) and PAI-1 secretion in the culture are modified by FSH, forskolin, dbcAMP, GnRH, PMA and growth factors (EGF and FGF), but not by hCG and androstenedione (△4); (iii) in vitro secretion of tPA and PA-PAI-1 complexes of Sertoli cells are greatly enhanced by presence of Leydig cells which produce negligible tPA but measurable PAI-1 activity;(iv) combination culture of Sertoli and Leydig cells remarkably increases FSH-induced PAI-1 activity and decreases hCG- and forskolin-induced inhibitor activity as compared with that of two cell types cultured alone. These data suggest that rat Sertoli cells, similar to ovarian granulosa cells, are capable of secreting both tPA and uPA, as well as PAI-1. The interaction of Sertoli cells and Leydig cells is essential for the cells to response to 相似文献
In the central nervous system, tissue plasminogen activator (tPA) plays a role in synaptic plasticity and remodeling. Our recent study has suggested that tPA participates in the rewarding effects of morphine by regulating dopamine release. In this study, we investigated the role of tPA in methamphetamine (METH)-related reward and sensitization. Repeated METH treatment dose-dependently induced tPA mRNA expression in the frontal cortex, nucleus accumbens, striatum and hippocampus, whereas single METH treatment did not affect tPA mRNA expression in these brain areas. The METH-induced increase in tPA mRNA expression in the nucleus accumbens was completely inhibited by pre-treatment with R(+)-SCH23390 and raclopride, dopamine D1 and D2 receptor antagonists, respectively. In addition, repeated METH treatment increased tPA activity in the nucleus accumbens. There was no difference in METH-induced hyperlocomotion between wild-type and tPA-deficient (tPA-/-) mice. On the other hand, METH-induced conditioned place preference and behavioral sensitization after repeated METH treatment were significantly reduced in tPA-/- mice compared with wild-type mice. The defect of behavioral sensitization in tPA-/- mice was reversed by microinjections of exogenous tPA into the nucleus accumbens. Our findings suggest that tPA is involved in the rewarding effects as well as the sensitization of the locomotor-stimulating effect of METH. 相似文献
Summary The major pol activity of CHO cells was purified 2 800-fold to near homogeneity and was characterized with respect to its physical and catalytic properties. The purified enzyme, upon analysis in denaturing activity gels, displayed a major, 120 kilodalton, catalytically active core and two minor, catalytically inactive components of 180 and 135 kilodaltons. The native form of the enzyme behaved in velocity sedimentation and gel permeation experiments as an asymmetric protein of an apparent Mr. of 515 kilodaltons. The purified enzyme displayed catalytic behavior and inhibitor sensitivity typical of that displayed by other mammalian pol alphas. Specifically, the enzyme: (1) was sensitive to n-ethylmaleimide and the pol -specific inhibitors, BuPdGTP and aphidicolin; (2) was subject to neutralization by specific monoclonal antibodies raised against human pol ; (3) was devoid of detectable 3 to 5 exonuclease activity, and (4) displayed a ribonucleotide-dependent DNA primase activity. 相似文献
mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator
inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts
grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene
plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown
that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic
activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was
due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575–582, 1986). The present study shows that there
is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates.
There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four
substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the
genetic level. 相似文献
Egg-laying hens are important candidate bioreactors for pharmaceutical protein production because of the amenability of their eggs for protein expression. In this study, we constructed an oviduct-specific vector containing tissue plasminogen activator (tPA) protein and green fluorescent protein (pL-2.8OVtPAGFP) and assessed its expression in vitro and in vivo. Oviduct epithelial and 3T3 cells were cultured and transfected with pL-2.8OVtPAGFP and pEGP-N1 (control vector), respectively. The pL-2.8OVtPAGFP vector was administered to laying hens via a wing vein and their eggs and tissues were examined for tPA expression. The oviduct-specific vector pL-2.8OVtPAGFP was expressed only in oviduct epithelial cells whereas pEGP-N1 was detected in oviduct epithelial and 3T3 cells. Western blotting detected a 89 kDa band corresponding to tPA in egg white and oviduct epithelial cells, thus confirming expression of the protein. The amount of tPAGFP in eggs ranged 9 to 41 ng/mL on the third day after vector injection. The tPA expressed in egg white and oviduct epithelial cells showed fibrinolytic activity, indicating that the protein was expressed in active form. GFP was observed only in oviducts, with no detection in heart, muscle, liver and intestine. This is the first study to report the expression of tPA in egg white and oviduct epithelial cells using an oviduct-specific vector. 相似文献
Ammonium ion concentrations ranging from 0 to 10 mM are shown to significantly reduce the sialylation of granuiocyte colony-stimulating factor (G-CSF) produced by recombinant Chinese hamster ovary cells. Specifically, the degree of completion of the final reaction in the O-linked glycosylation pathway, the addition of sialic acid in an alpha(2,6) linkage to N-acetylgalactosamine, is reduced by NH(4) (+) concentrations of as low as 2 mM. The effect of ammonia on sialylation is rapid, sustained, and does not affect the secretion rate of G-CSF. Additionally, the effect can be mimicked using the weak base chloroquine, suggesting that the effect is related to the weak base characteristics of ammonia. In support of this hypothesis, experiments using brefeldin A suggest that the addition of sialic acid in an alpha(2,6) linkage to N-acetylgalactosamine occurs in the trans-Golgi compartment prior to the trans-Golgi network, which would be expected under normal conditions to have a slightly acidic pH in the range from 6.5 to 6.75. Ammonium ion concentrations of 10 mM would be expected to reduce significantly the differences in pH between acidic intracellular compartments and the cytoplasm. The pH-activity profile for the CHO O-linked alpha(2,6) sialytransferase using monosialylated G-CSF as a substrate reveals a twofold decrease in enzymatic activity across the pH range from 6.75 to 7.0.Mathematical modeling of this sialylation reaction supports the hypothesis that this twofold decrease in sialyltransferase activity resulting from an ammoniainduced increase in trans-Golgi pH could produce the observed decrease in G-CSF sialylation. (c) 1995 John Wiley & Sons, Inc. 相似文献
Alcohol exposure affects neuronal plasticity in the adult and developing brain. Astrocytes play a major role in modulating neuronal plasticity and are a target of ethanol. Tissue plasminogen activator (tPA) is involved in modulating neuronal plasticity by degrading the extracellular matrix proteins including fibronectin and laminin and is up‐regulated by ethanol in vivo. In this study we explored the hypothesis that ethanol affects DNA methylation in astrocytes thereby increasing expression and release of tPA. It was found that ethanol increased tPA mRNA levels, an effect mimicked by an inhibitor of DNA methyltransferase (DNMT) activity. Ethanol also increased tPA protein expression and release, and inhibited DNMT activity with a corresponding decrease in DNA methylation levels of the tPA promoter. Furthermore, it was observed that protein levels of DNMT3A, but not DNMT1, were reduced in astrocytes after ethanol exposure. These novel studies show that ethanol inhibits DNA methylation in astrocytes leading to increased tPA expression and release; this effect may be involved in astrocyte‐mediated inhibition of neuronal plasticity by alcohol.
Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cdr2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd2+ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd2+-resistant Cdr2C10 subline to induce synthesis of the Cd2+- and Zn2+-binding protein(s), metallothionein(s) (MT). Evidence that Cd2+ behaves as an analog of the essential trace metal, Zn2+, especially as an inducer of MT synthesis, suggested that the Cdr and CHO cell types could be employed to investigate cellular Zn2+ metabolism. In the present study, measurements were made to compare CHO and Cdr cell types for (a) growth as a function of the level of ZnCl2 added to the culture medium, (b) uptake and subcellular distribution of Zn2+, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cdr cell types grew normally (Td≊16–18 h) during exposures to Zn2+ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn2+ levels, (b) Cdr cells incorporate fourfold more Zn2+ during a 24-h exposure to the maximal subtoxic level of Zn2+ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cdr cell is proficient in this response during exposure to the maximal subtoxic Zn2+ level. These findings suggest that (a) the CHO and Cdr cell systems will be useful in further studies of cellular Zn2+ metabolism, especially in comparisons of Zn2+ metabolism in the presence and absence of induction of the Zn2+-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular
Zn2+ uptake. 相似文献
Summary The mesothelial cells obtained from human omental adipose tissue showed a typical cobblestone monolayer and reacted strongly
with keratin, but did not have Von Willebrand factor. Ultrastructurally these cells revealed the existence of desmosome-like
cell junctions as well as intracellular canaliculi, tubular structures surrounded by microvilli, and tonofilament-like filaments.
The mesothelial cells grew much faster in the medium containing epidermal growth factor, actively took up acetylated-low density
lipoprotein into their cytoplasm, and released angiotensin-converting enzyme. They also released urokinase-type plasminogen
activator, but only half as much as do human umbilical vein endothelial cells; release of tissue-type plasminogen activator
was not observed. Inasmuch as the mesothelial cells also released plasminogen activator inhibitor-1, as do human umbilical
vein endothelial cells, we could not detect u-PA activity in culture medium. u-PA may play a role in the protection against
adhesion among visceral organs. These observations indicate that cultured human mesothelial cells have characteristics closely
related to those found in human endothelial cells. 相似文献
We have previously demonstrated that repeated, but not acute, methamphetamine (METH) treatment increases tissue plasminogen activator (tPA) activity in the brain, which is associated with the development of behavioral sensitization to METH. In this study, we investigated whether the tPA-plasmin system is involved in the development of sensitization in METH-induced dopamine release in the nucleus accumbens (NAc). There was no difference in acute METH-induced increase in extracellular dopamine levels in the NAc between wild-type and tPA-deficient (tPA−/−) mice. Repeated METH treatment resulted in a significant enhancement of METH- induced dopamine release in wild-type mice, but not tPA−/− mice. Microinjection of exogenous tPA or plasmin into the NAc of wild-type mice significantly potentiated acute METH- induced dopamine release. Degradation of laminin was evident in brain tissues incubated with tPA plus plasminogen or plasmin in vitro although tPA or plasminogen alone had no effect. Immunohistochemical analysis revealed that microinjection of plasmin into the NAc reduced laminin immunoreactivity without neuronal damage. Our findings suggest that the tPA-plasmin system participates in the development of behavioral sensitization induced by repeated METH treatment, by regulating the processes underlying the sensitization of METH-induced dopamine release in the NAc, in which degradation of laminin by plasmin may play a role. 相似文献
When parental Chinese hamster ovary (CHO) cell clones that are capable of producing thrombopoietin (TPO) were subjected to
high methotrexate (MTX) concentrations, clonal variations in cell growth were apparent. In the clones that had no significant
enhancement in specific TPO productivity (qTpo) when a higher level of MTX was administered, their growth was not depressed significantly nor their cell size changed significantly.
On the other hand, those clones that showed a significant enhancement inqTpo at higher a MTX dosage, cell growth was depressed initially but recovered during successive sub-cultures. Furthermore, their
cell size increased, which suggested that changes in cell size may be indicative of an enhancedqTpo. When the enhancement of theqTpo of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation
(r2=0.80,P<0.001, ANOVA), which suggested that an enhancement ofqTpo after high MTX administration can be measured by the increase in their cell size. Taken together, our data demonstrate that
the selection of amplified CHO cell clones with enhancedqTpo can be done based upon their increased size and growth pattern. This facilitates the development of highly productive recombinant
CHO cell lines. 相似文献
The extracellular serine protease tissue plasminogen activator (tPA) that converts plasminogen into plasmin is abundantly expressed throughout the central nervous system. We have recently demonstrated that the tPA-plasmin system participates in the rewarding and locomotor-stimulating effects of morphine by acutely regulating morphine-induced dopamine release in the nucleus accumbens (NAc). In the present study, we examined the effects of microinjections of plasminogen activator inhibitor-1 (PAI-1), tPA or plasmin into the NAc on morphine-induced dopamine release, hyperlocomotion and anti-nociceptive effects in ICR mice. A single morphine treatment resulted in an increase in protein levels of PAI-1 in the NAc. Microinjection of PAI-1 into the NAc dose-dependently reduced morphine-induced dopamine release and hyperlocomotion. In contrast, microinjection of tPA into the NAc significantly potentiated morphine-induced dopamine release and hyperlocomotion without affecting basal levels. Furthermore, microinjection of plasmin enhanced morphine-induced dopamine release, but did not modify the hyperlocomotion induced by morphine. The intracerebroventricular injection of PAI-1, tPA and plasmin at high doses had no effect on the anti-nociceptive effects of morphine. These results suggest that the tPA-plasmin system is involved in the regulation of morphine-induced dopamine release and dopamine-dependent behaviors but not the anti-nociceptive effects of morphine. 相似文献
Pituitary adenylate cyclase-activating polypeptide (PACAP) and tissue plasminogen activator (tPA) play important roles in neuronal migration and survival. However, a direct link between the neurotrophic effects of PACAP and tPA has never been investigated. In this study, we show that, in PC12 cells, PACAP induced a 9.85-fold increase in tPA gene expression through activation of the protein kinase A- and protein kinase C-dependent signaling pathways. In immature cerebellar granule neurons (CGN), PACAP stimulated tPA mRNA expression and release of proteolytically active tPA. Immunocytochemical labeling revealed the presence of tPA in the cytoplasm and processes of cultured CGN. The inhibitory effect of PACAP on CGN motility was not affected by the tPA substrate plasminogen or the tPA inhibitor plasminogen activator inhibitor-1. In contrast, plasminogen activator inhibitor-1 significantly reduced the stimulatory effect of PACAP on CGN survival. Altogether, these data indicate that tPA gene expression is activated by PACAP in both tumoral and normal neuronal cells. The present study also demonstrates that PACAP stimulates the release of tPA which promotes CGN survival by a mechanism dependent of its proteolytic activity. 相似文献