One important limitation of the widely used insect baculovirus overexpression system is its inefficiency to properly process heterologous proteins which are initially biosynthesized as larger ...inactive precursor proteins. One example is transforming growth factor beta 1 (TGF beta 1), a 25-kDa homodimeric protein with pleiotropic functions. As many growth factors, the inactive TGF beta 1 precursor molecule needs to be proteolytically cleaved C-terminal to a basic sequence to yield the mature and active homodimer. In insect cells, a large proportion of overexpressed TGF beta 1 was found in an inactive precursor form suggesting that the levels of endogenous convertases are limiting for the production of mature and bioactive TGF beta 1 in this system. We have demonstrated that furin, a member of a novel family of mammalian prohormone convertases (PCs) can efficiently process TGF beta 1 precursor resulting in the production of the mature and active growth factor. Taking advantage of this observation, we have developed an improved overproduction system for TGF beta 1 by coexpressing prohTGF beta 1 and human furin convertase in High Five cells. Using this system, the production of mature active TGF beta 1 increased in a dose-dependent fashion reaching up to 7.8-fold the amount obtained with the growth factor only. Thus, eliminating the rate-limiting step in recombinant TGF beta 1 production maximizes its processing efficiency and the yield of the mature active growth factor. Such simple and efficient technology could be useful for large scale production of other proproteins which undergo similar maturation processes and share furin recognition sequences at the junction between the proregion and the mature polypeptide.
It is well known that the adrenal zona glomerulosa is transformed to zona fasciculata-reticularis in rats exposed chronically to ACTH. This model was used to study the intracellular distribution of ...protein kinase C, which is known to be involved in differentiation processes. Under basal conditions, in zona glomerulosa, 70, 23, and 7% of the protein kinase C was located in the cytosol, membrane and nuclear fractions, respectively. At 30 min after ACTH administration to rats, the protein kinase C content remained unchanged in the nuclear fraction, whereas that of the cytosolic fraction was decreased to 43% while in the membranes it was increased to 48%. After 2 days of ACTH treatment, we observed a significant increase, up to 25%, of protein kinase C in the nuclear fraction, a decrease to 47% in the cytosol, whereas the membrane fraction content had returned to its basal value. The intracellular distribution of inner zones was 17% in nuclear fraction, 47% in cytosol and 36% in membranes. ACTH treatments did not change these proportions. The total protein kinase C content of ACTH-treated groups was not different than that of their respective controls, in zona glomerulosa and in inner zones respectively. The cytosolic protein kinase C formed complexes with detergent-treated nuclei; this association was saturable, and could be measured by the ability of the kinase to bind 3HPDBu (20(n)-3Hphorbol-12,13-dibutyrate). The number of nuclear 'acceptor sites' thus measured was calculated to be 5245 fmol/mg DNA in the zona glomerulosa; this did not change significantly following a 3-day administration of ACTH. Protein kinase C prepared from the adrenal inner zones also bound zona glomerulosa detergent-treated nuclei but occupied fewer sites than the protein kinase C from the zona glomerulosa. In conclusion, the effects of chronic ACTH treatment on rat adrenal zona glomerulosa could be mediated by an increased level of protein kinase C in the nuclear fraction and possibly through its binding to specific 'acceptor sites'.
We investigated the application of octadecylsilyl (ODS)-silica in studies related to characterization and purification of the nontransformed rat ventral prostate androgen receptor. The results ...indicated that ODS-silica successfully separates the free 3HR1881 from the labeled 3HR1881 transformed (4-5S) and nontransformed (8-9S) rat ventral prostate androgen receptors. Partial purification of the 8-9S receptor form was performed by the fractionation of rat cytosol using cartridges of the ODS-silica and fast-flow-rate phosphocellulose chromatography. Further purification was accomplished by differential chromatography (DEAE-cellulose and slow-flow-rate phosphocellulose chromatography). This partially purified 8-9S receptor, when analyzed on a gel permeation high-performance liquid chromatography column, resulted in a three-peak pattern of UV absorbance. One of these peaks corresponded to a 59-kD non3HR1881-binding protein and the remaining two corresponded to 270-kD and 190-kD3HR1881-binding proteins. These results demonstrate the usefulness of ODS-silica in androgen receptor studies. The association of a 59-kD nonsteroid binding protein with the nontransformed rat ventral prostate androgen receptor is discussed.
The present study reports a 3,800‐fold purification of the 8‐9S androgen‐receptor complex from benign prostate hyperplasia (BPH) tissues using differential chromatography. In addition, the BPH ...androgen receptor complexes have been characterized using sucrose density gradient (SDG) ultracentrifugation, gel permeation, and anion exchange high performance liquid chromatography (HPLC). Results indicate that a) under nontransforming conditions, BPH cytosols contained both 8‐9S (40–78%) and 4S (22–60%) androgen‐receptor forms, b) apparent molecular weights of these androgen‐receptor complexes, as analyzed by gel permeation HPLC, were estimated to correspond at 270 kDa, and 90 kDa respectively, c) 8‐9S androgen‐receptor complexes were retained on an anion exchange HPLC column and could be eluted at 0.22 M KCl at a linear gradient, whereas 4S complexes were not retained on anion exchange columns under identical experimental conditions, d) 10X dilution of BPH cytosols containing only the 4S (0.6 M KCl) form and subsequent chromatography on anion exchange HPLC system was indicative of fragmentation (these fragments were retained on anion exchange columns and could be eluted by 0.33 M KCl on a linear gradient HPLC), and e) increased temperature (22 C) was permissive of proteolytic fragmentation (fragments were estimated to correspond at 30, 15, and 5 kDa). The results are discussed in relationship with the composition of the nontransformed androgen‐receptor molecules.
Tri-
n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-
n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes ...through pathways involving the endoplasmic reticulum and mitochondria. Tri-
n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri-
n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad from the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri-
n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri-
n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri-
n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl
−/HCO
3
− exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri-
n-butyltin-induced apoptotic signaling in rat hepatocytes.
Hepatocytes are an important physiological model for in vitro studies of drug metabolism and toxicity. However, fresh hepatocytes are not always available and hence cyopreservation is needed to ...preserve large quantities until they are needed for these applications. Hepatocytes are extremely sensitive to damage induced by the freeze-thaw process, even after addition of traditional cryoprotectants such as dimethyl sulfoxide (DMSO). Furthermore, they do not proliferate in culture. We previously demonstrated that a crude wheat extract protects rat hepatocytes during cryopreservation and could provide a promising alternative to DMSO. We have considerably improved this novel cryopreservation procedure by using wheat extracts that are partially purified by either ammonium sulphate or acetone precipitation, or by using recombinant wheat freezing tolerance-associated proteins such as WCS120, TaTIL, WCS19, and TaIRI-2. These improved procedures enhance long-term storage (2-12 months) and recovery of large quantities of healthy cells after cryopreservation, and maintain the differentiated functions of rat hepatocytes, compared to freshly isolated cells, as judged by viability (77-93%), adherence (77%) and metabolic functions of major cytochrome P450 isoforms CYP1A1/2, CYP2C6, CYP2D2, and CYP3A1/2. The advantage of using wheat proteins as cryopreservants is that they are non-toxic, natural products that do not require animal serum, and are economical and easy to prepare. Biotechnol. Bioeng. 2009;103: 582-591.
Autophagy, an essential intracellular recycling process, is linked to the pathogenesis of various diseases including Crohn’s disease (CD). Factors that lead to the development of impaired autophagy ...during intestinal inflammation remain largely unexplored. Here, we report the impact of the interaction between serotonin 5-hydroxytryptamine;(5-HT) and autophagy in colitis in mouse and human studies. In mice, increased gut 5-HT inhibited autophagy and led to enhanced colitis susceptibility. Reciprocally, mice with reduced 5-HT exhibited up-regulated autophagy via the mammalian target of rapamycin pathway, which resulted in significantly decreased colitis. Deletion of autophagy gene, Atg7, in an epithelial-specific manner, in concert with reduced 5-HT, promoted the development of a colitogenic microbiota and abolished the protective effects conferred by reduced 5-HT. Notably, in control and patient peripheral blood mononuclear cells, we uncovered that 5-HT treatment inhibited autophagy. Our findings suggest 5-HT as a previously unidentified therapeutic target in intestinal inflammatory disorders such as CD that exhibits dysregulated autophagy.
Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces ...viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell–cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (β1-integrin, E-cadherin, and β-catenin). Immunoblot analyses revealed that the levels of β1-integrin, E-cadherin, and β-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for β1-integrin (62–74%) > β-catenin (51–58%) > E-cadherin (21–37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of β1-integrin, E-cadherin, and β-catenin. Protein expression was only 10–20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good expression of these adhesion molecules. These promising results could lead to a new and improved cryopreservation technology for applications such as clinical transplantation of hepatocytes.
The drug discovery and development process requires adequate safety testing for drug toxicity before new drugs can be administered to patients. Hepatocytes are used in vitro to screen compounds for ...hepatotoxicity, induction of drug-metabolizing enzymes such as cytochrome P450 (P450) isoforms, drug-drug interactions, and establish human relevance for metabolism. Cryopreservation makes it possible to preserve a large quantity of functional hepatocytes. Techniques for cryopreservation of hepatocytes are mainly based on dimethyl sulfoxide (DMSO). However, analyses of metabolic capacities of cryopreserved hepatocytes are often limited by loss of functional integrity of hepatocytes after thawing. Therefore, it is necessary to improve techniques of cryopreservation. We have developed a new cryopreservation technology for mammalian cells based on a wheat protein extract (WPE). We determined whether the WPE can better preserve activities of major P450 isoforms both in suspension and monolayer cultures of hepatocytes. This was achieved by comparing basal and inducible or metabolic activities of isoforms CYP1A1, CYP1A2, CYP2C6, CYP2D2, and CYP3A in rat hepatocytes that were cryopreserved with WPE, relative to fresh cells and those cryopreserved with DMSO. We conclusively show that rat hepatocytes cryopreserved with WPE retain their metabolic competency and their ability to respond to classical P450 inducers when compared with freshly isolated hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for rat hepatocytes. They are an efficient, nontoxic, economic natural product and universal cryoprotectant that is superior to DMSO, which has limitations because of cellular toxicity.
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