Refolding and purification of recombinant human PDE7A expressed in Escherichia coli as inclusion bodies

We have investigated the refolding and purification of the catalytic domain of human 3',5'-cyclic nucleotide phosphodiesterase 7A1 (PDE7A1) expressed in Escherichia coli. A cDNA encoding an N-terminal-truncated PDE7A1(147-482-His) was amplified by RT-PCR from human peripheral blood cells and inserted into the vector pET21-C for bacterial expression of the enzyme fused to a C-terminal His-tag. The PDE was found to be expressed in the form of inclusion bodies which could be refolded to an active enzyme in buffer containing high concentrations of arginine hydrochloride, ethylene glycol, and magnesium chloride at pH 8.5. The PDE7A1(147-482-His) construct could be purified after dialysis and concentration steps by either Zn2+-IDA-Sepharose chromatography or ResourceQ ion-exchange chromatography to homogeneity. In comparison to the metal-chelate column, the ResourceQ purification resulted in a distinctly better yield and enrichment of the protein. Both the Vmax (0.46 micromol. min(-1). mg(-1) ) and the K(m) (0.1 microM) of the purified enzyme were found to be comparable with published data for native or recombinant catalytically active expressed PDE7A1. Using SDS/PAGE, a molecular mass of 39 kDa was determined (theoretical value 38.783 kDa). As known from several other mammalian PDEs, size-exclusion chromatography using refolded PDE7A1(147-482-His) indicated the formation of dimers. The purified enzyme was soluble at concentrations up to 100 microg/ml. A further increase of protein concentration resulted, however, in precipitation of the enzyme.     

Regulation of the cell cycle of B-lymphocytes in mice by substances elevating the levels of intracellular cAMP and cGMP

Spontaneous velocity sedimentation of B lymphocytes activated by intraperitoneal injection of ovalbumin into mice was used to obtain cell cycle synchronized cells, evidenced by differences in the incorporation of labeled precursors of protein and nucleic synthesis (14C-methionine and 3H-thymidine). The effects of acetylcholine and adrenaline, cAMP and cGMP on the intensity of 3H-thymidine incorporation into mouse B lymphocytes and on the amount of the cells entering mitosis were examined. It was shown that acetylcholine is capable of stimulating whereas adrenaline of inhibitin B lymphocyte entry into the stage of DNA synthesis and egress of these cells from the stage of DNA synthesis to the stage of mitosis. Adrenaline was found to have a reciprocal action. The acetylcholine effect could be mimetized by exogenous cGMP, that of adrenaline by cAMP. Stimulation of the G1/S transition was mediated by intracellular calcium ions but did not depend on exocellular calcium.     

Parallel Allostery by cAMP and PDE Coordinates Activation and Termination Phases in cAMP Signaling

The second messenger molecule cAMP regulates the activation phase of the cAMP signaling pathway through high-affinity interactions with the cytosolic cAMP receptor, the protein kinase A regulatory subunit (PKAR). Phosphodiesterases (PDEs) are enzymes responsible for catalyzing hydrolysis of cAMP to 5′ AMP. It was recently shown that PDEs interact with PKAR to initiate the termination phase of the cAMP signaling pathway. While the steps in the activation phase are well understood, steps in the termination pathway are unknown. Specifically, the binding and allosteric networks that regulate the dynamic interplay between PKAR, PDE, and cAMP are unclear. In this study, PKAR and PDE from Dictyostelium discoideum (R_D and RegA, respectively) were used as a model system to monitor complex formation in the presence and absence of cAMP. Amide hydrogen/deuterium exchange mass spectrometry was used to monitor slow conformational transitions in R_D, using disordered regions as conformational probes. Our results reveal that R_D regulates its interactions with cAMP and RegA at distinct loci by undergoing slow conformational transitions between two metastable states. In the presence of cAMP, R_D and RegA form a stable ternary complex, while in the absence of cAMP they maintain transient interactions. RegA and cAMP each bind at orthogonal sites on R_D with resultant contrasting effects on its dynamics through parallel allosteric relays at multiple important loci. R_D thus serves as an integrative node in cAMP termination by coordinating multiple allosteric relays and governing the output signal response.     

Zyxin is up-regulated during megakaryocytic differentiation of human UT-7/c-mpl cells

To characterize genes involved in megakaryocytic commitment, we compared expression profiles of bipotent cells (UT-7/c-mpl) with those of the same cells induced to differentiate towards megakaryopoiesis in the presence of TPO. Using cDNA arrays, we showed that 12 out of 2260 genes changed their expression level after 6h of TPO stimulation. One of these genes encodes for zyxin, a cytoskeleton protein component. Zyxin is up-regulated at the mRNA and protein levels in UT-7/c-mpl cells in response to TPO confirming the reliability of the cDNA array technology. Similarly, when CD34 positive cells were induced to differentiate into megakaryocytes, zyxin mRNA was accumulated. Furthermore, when megakaryocytes were allowed to spread on fibrinogen, formation of stress fibers and lamellipodia was induced and zyxin was localized at the picks of actin stress fibers. These results suggest an important role for zyxin during megakaryocytic differentiation and more precisely in the regulation of the integrin mediated adhesion process in megakaryocytes.     

Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP.

Early passaged bovine pulmonary artery smooth muscle cells (SMC) respond to serotonin (5-HT) by developing a reversible change in configuration. (Lee et al. J. Cell. Physiol. 138:145, 1989). This configurational change does not occur in pulmonary artery endothelial cells (EC) subjected to 5-HT and is adenosine triphosphate (ATP) dependent, lost with passage of SMC, and inhibited by various agents that block high-affinity 5-HT uptake. We now report a second configurational change (also dendritic formation) of SMC produced by 5-HT only in the presence of isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase. This configurational change was also ATP dependent, but unlike the first response, (Lee et al., 1989), it occurred in both first and later passaged SMC and was not inhibited by blockade of 5-HT uptake. Also, unlike the response with 5-HT alone that failed to elevate cAMP, this one was associated with a large elevation of cAMP (eight fold above control values), similar to the response to the beta-agonist isoproterenol, plus IBMX. The second response was not blocked by a variety of 5-HT receptor antagonists but was reproduced by (+/-)-8-hydroxy-DPAT HBr (8-OH-DPAT), a reputed 5-HT1A agonist. The response was not dependent upon Ca2+ and was blocked by 1-2 mM n-phenylanthranilic acid or anthracene-9-carboxylic acid, electrically conductive Cl- channel inhibitors. Hence, 5-HT in the presence of IBMX causes a marked elevation of cAMP of SMC and this elevation in cAMP likely results in a cellular configurational change through a Cl- channel-dependent mechanism similar to that we previously described for EC in the presence of beta-adrenergic agonist stimulation (Ueda et al. Circ. Res. 66:951, 1990). EC do not show a similar response to 5-HT possibly because cAMP is not adequately elevated, even in the presence of IBMX, to enhance Cl- channel activity. We propose that our observations indicate the presence of two sites of action of 5-HT on the smooth muscle cell, one intracellularly and another at a cell surface receptor.     

VAMP2 is expressed in muscle satellite cells and up-regulated during muscle regeneration

Membrane trafficking is one of the most important mechanisms involved in the establishment and maintenance of the forms and functions of the cell. However, it is poorly understood in skeletal muscle cells. In this study, we have focused on vesicle-associated membrane proteins (VAMPs), which are components of the vesicle docking and fusion complex, and have performed immunostaining to investigate the expression of VAMPs in rat skeletal muscle tissue. We have found that VAMP2, but not VAMP1 or VAMP3, is expressed in satellite cells. VAMP2 is also expressed in myofibers in the soleus muscle and nerve endings. This is consistent with previous studies in which VAMP2 has been shown to regulate GLUT4 trafficking in slow-twitch myofibers in soleus muscle and neurotransmitter release in nerve endings. As satellite cells are quiescent myogenic cells, the expression of VAMP2 has further been examined in regenerating muscles after injury by the snake venom, cardiotoxin; we have observed enhanced expression of VAMP2 in immature myotubes with a peak at 3 days after injury. Our findings suggest that VAMP2 plays roles in quiescent satellite cells and is involved in muscle regeneration. The nature of the material transported in the VAMP2-bearing vesicles in satellite cells and myotubes is still under investigation. This work was supported by a research grant (17A-10) for nervous and mental disorders from the Ministry of Health, Labor, and Welfare of Japan, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

Purification and characterization of the human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells

The human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells was found to be heavily phosphorylated on both serines of the conserved SPS motif by mass spectrometric analysis. The purified protein exists as a tetramer at a concentration approximately 1 mg/ml from light scattering measurement and has a Km of 2 microM in hydrolyzing cAMP. In comparison, a partially purified PDE4A330-723 expressed in Escherichia coli has an apparent Km of 10 microM. The EC50 values for the Mg2+- or Co2+-mediated cAMP hydrolysis between the two enzymes differed by less than twofold. In addition, both enzymes exhibit similar sensitivities toward inhibition by a diverse set of inhibitors. Together with the fact that its adjacent peptide was covalently labeled by an electrophilic cAMP analogue, these results support that the SPS motif is not part of but is positioned near the active site. An efficient purification protocol that provides a highly purified PDE4A catalytic domain suitable for crystallization study is described.     

The nifk gene is widely expressed in mouse tissues and is up-regulated in denervated hind limb muscle

Denervation of skeletal muscle alters the expression of many genes, which may be important for establishing optimal conditions for reinnervation. Using the differential display technique we have attempted to discover neurally regulated genes in skeletal muscle. An mRNA that is up-regulated in denervated hind limb muscle was identified and cloned. The cDNA encodes an RNA-binding protein, which was discovered during the course of this work to be a nucleolar protein interacting with the fork-head associated domain of the proliferation marker protein Ki-67, and named NIFK. We show that the nifk gene is widely expressed in adult mouse tissues and that the expression is up-regulated in denervated hind limb muscle. No difference between expression in perisynaptic and extrasynaptic portions of muscle was observed. The widespread expression in adult tissues suggests that the NIFK protein has other functions in addition to its interaction with Ki-67, which is only expressed in proliferating cells.     

SLC26A7 is a Cl- channel regulated by intracellular pH

Members of the SLC26 transporter family play an essential role in several epithelial functions, as revealed by diseases associated with mutations in members of the family. Several members were shown to function as Cl(-) and HCO(3)(-) transporters that likely play an important role in epithelial Cl(-) absorption and HCO(3)(-) secretion. However, the mechanism of most transporters is not well understood. SLC26A7 is a member of the SLC26 transporter family reported to be expressed in the basolateral membrane of the cortical collecting duct and parietal cells and functions as a coupled Cl(-)/HCO(3)(-) exchanger. In the present work we examined the transport properties of SLC26A7 to determine its transport characteristics and electrogenicity. We found that when expressed in Xenopus oocytes or HEK293 cells SLC26A7 functions as a pH(i)-regulated Cl(-) channel with minimal OH(-)/HCO(3)(-) permeability. Expression of SLC26A7 in oocytes or HEK293 cells generated a Cl(-) current with linear I/V and an instantaneous current that was voltage- and time-independent. Based on measurement of reversal potential the selectivity of SLC26A7 is NO(3)(-)>Cl(-)=Br(-)=I(-)>SO(4)(2-)=Glu(-), although I(-) partially inhibited the current. Incubating the cells with HCO(3)(-) or butyrate acidified the cytosol and increased the selectivity of SLC26A7 for Cl(-). Measurement of membrane potential and pH(i) showed minimal OH(-) and HCO(3)(-) transport by SLC26A7 when the cells were incubated in Cl(-)-containing or Cl(-)-free media. The activity of SLC26A7 was inhibited by all inhibitors of anion transporters tested, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylic acid, and glybenclamide. These findings reveal that SLC26A7 functions as a unique Cl(-) channel that is regulated by intracellular H(+).     

Group IID heparin-binding secretory phospholipase A(2) is expressed in human colon carcinoma cells and human mast cells and up-regulated in mouse inflammatory tissues.

Group IID secretory phospholipase A(2) (sPLA(2)-IID), a heparin-binding sPLA(2) that is closely related to sPLA(2)-IIA, augments stimulus-induced cellular arachidonate release in a manner similar to sPLA(2)-IIA. Here we identified the residues of sPLA(2)-IID that are responsible for heparanoid binding, are and therefore essential for cellular function. Mutating four cationic residues in the C-terminal portion of sPLA(2)-IID resulted in abolition of its ability to associate with cell surface heparan sulfate and to enhance stimulus-induced delayed arachidonate release, cyclooxygenase-2 induction, and prostaglandin generation in 293 cell transfectants. As compared with several other group II subfamily sPLA(2)s, which were equally active on A23187- and IL-1-primed cellular membranes, sPLA(2)-IID showed apparent preference for A23187-primed membranes. Several human colon carcinoma cell lines expressed sPLA(2)-IID and sPLA(2)-X constitutively, the former of which was negatively regulated by IL-1. sPLA(2)-IID, but not other sPLA(2) isozymes, was expressed in human cord blood-derived mast cells. The expression of sPLA(2)-IID was significantly altered in several tissues of mice with experimental inflammation. These results indicate that sPLA(2)-IID may be involved in inflammation in cell- and tissue-specific manners under particular conditions.     

Epithelial permeability induced by neutrophil transmigration is potentiated by hypoxia: Role of intracellular cAMP

Mucosal tissues, such as the lung and intestine, are primary targets for ischemic damage. Under these conditions, neutrophil (polymorphonuclear leukocyte; PMN) infiltration into the protective epithelium has been implicated as a pathophysiologic mediator. Because PMN transepithelial migration results in increased paracellular permeability, and because our previous data revealed that epithelial hypoxia enhances PMN transmigration, we hypothesized that macromolecular permeability may be altered in epithelium exposed to hypoxia and reoxygenation (H/R) in the presence of PMNs. Human intestinal epithelia (T84) were grown on permeable supports, exposed to cellular hypoxia (pO₂ 20 torr) for 0–72 hr, and examined for increases in PMN-evoked permeability by using standard flux assays. Increasing epithelial hypoxia potentiated PMN-induced permeability of labeled paracellular tracers (size range 3–500 kD). Such increases were blocked by monoclonal antibody (mAb) to the PMN integrin CD11b (82 ± 1% decreased compared with control mAb) and were partially blocked by anti-CD47 mAb(51 ± 1%). Assessment of barrier recovery revealed that monolayers exposed to H/R were significantly diminished in their ability to reseal following PMN transmigration (recovery of 36 ± 6% in H/R vs. 94 ± 2% in normoxic controls). Because intracellular cyclic AMP (cAMP) has been demonstrated to regulate epithelial permeability, and because PMN-derived compound(s), (i.e., 5′-adenosine monophosphate; AMP) elevate epithelial cAMP, we examined the impact of hypoxia on epithelial cAMP responses. These experiments revealed that hypoxic epithelia were diminished in their ability to generate cAMP, and pharmacologic elevation (8-bromo-cAMP) of intracellular cAMP in hypoxic cells normalized both PMN-induced permeability changes and restoration of barrier function. These results support a role for PMN in increased intestinal permeability associated with reperfusion injury and imply a substantial role for cAMP signaling in maintenance of permeability during PMN transmigration. J. Cell. Physiol. 176:76–84, 1998. © 1998 Wiley-Liss, Inc.     

Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli

A 5'-truncated PDE4A-cDNA corresponding to the amino acid positions 200-886 of the "full-length" sequence (Accession No. L20965) was generated from human leukocyte mRNA by RT-PCR. Several PDE4A constructs containing the catalytic region and differing in their degree of N- and/or C-terminal truncation (amino acid positions 200-886, 200-704, 342-886, and 342-704) were expressed in Escherichia coli to investigate the effect of truncations on purification characteristics, long-term stability, and aggregation. All peptides accumulated as inclusion bodies, necessitating refolding prior to purification by dye and metal chelate affinity chromatography. The constructs differed in long-term stability due to variable levels of protease contamination. The position of the His-tag also influenced the purification results. The best results were obtained with the N- and C-truncated form C-terminally His-tagged, appropriate quantities of which were obtained in pure form and was found to be stable against proteolysis at 4 degrees C for at least 6 weeks. The comparison of the molecular mass of the investigated PDE4A constructs obtained by SDS electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation indicated that C-terminal truncated PDE4A forms dimers whereas PDE4A constructs with a complete C-terminus tend to form larger aggregates.     

CXCR7 protein is not expressed on human or mouse leukocytes

Since the discovery that CXCR7 binds to CXCL12/SDF-1α, the role of CXCR7 in CXCL12-mediated biological processes has been under intensive scrutiny. However, there is no consensus in the literature on the expression of CXCR7 protein by peripheral blood cells. In this study we analyzed human and mouse leukocytes and erythrocytes for CXCR7 protein expression, using a competitive CXCL12 binding assay as well as by flow cytometry and immunohistochemistry using multiple CXCR7 Abs. CXCR7(-/-) mice were used as negative controls. Together, these methods indicate that CXCR7 protein is not expressed by human peripheral blood T cells, B cells, NK cells, or monocytes, or by mouse peripheral blood leukocytes. CXCR7 protein is, however, expressed on mouse primitive erythroid cells, which supply oxygen to the embryo during early stages of development. These studies therefore suggest that, whereas CXCR7 protein is expressed by primitive RBCs during murine embryonic development, in adult mammals CXCR7 protein is not expressed by normal peripheral blood cells.     

Hepoxilin signaling in intact human neutrophils: biphasic elevation of intracellular calcium by unesterified hepoxilin A3

We have previously shown that the methyl ester of hepoxilin A3 causes a receptor-induced rise in intracellular calcium through the release from intracellular stores in suspended human neutrophils. The corresponding free acid was devoid of activity. We now report that the action of the free acid form of hepoxilin A3 is dependent on the type of vehicle used, i.e. it is active in releasing calcium when used in an ethanol vehicle but not in DMSO. The methyl ester is equally active in either vehicle. The pattern of calcium release between the free acid and the methyl ester is qualitatively different. Both compounds show a biphasic pattern, i.e. an initial rapid phase followed by a slow decline in calcium levels but never reaching pre-hepoxilin A3 baseline levels. The methyl ester appears slightly more potent in the initial phase of calcium release than the free acid (methyl = 188+/-14 S.D., free acid = 135+/-11 S.D. nM, P < 0.0005). Both compounds appear to reach the same calcium levels at the plateau of the second prolonged phase (methyl = 88+/-8 S.D., free acid = 107+/-15 S.D. nM, not significant). Lanthanum chloride (an inhibitor of calcium influx) interfered with the second phase of the curve causing calcium levels to return to normal pre-hepoxilin levels for both compounds. Addition of lanthanum chloride prior to the hepoxilin addition or carrying out the experiments in calcium-free medium, eliminated the second phase completely, with the calcium peak returning rapidly to normal baseline levels, suggesting that the second phase is due to calcium influx. Again the methyl ester is more active than the free acid (methyl, 189+/-12; free acid, 145+/-6 S.D. nM, P<0.005). Additional experiments with tritium-labelled methyl ester of hepoxilin A3 demonstrated that the compound is hydrolyzed into the free acid intracellularly. These experiments demonstrate that DMSO interacts with hepoxilin free acid, interfering with its entry into the cell while ethanol does not. Once inside the cell, hepoxilin interacts with its own receptor to release calcium rapidly from stores, but it also causes a more prolonged influx of calcium from the extracellular milieu.     

B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis

Human endothelial cells (ECs) provide costimulatory signals sufficient to activate resting memory T cells to produce IL-2 and IFN-gamma, at least in part through CD58-CD2 interactions. Recently, the B7-like molecule, B7-H1 (PD-L1), was described and shown to regulate T cell activation; however, there are conflicting reports on whether it stimulates or inhibits T cell cytokine synthesis. B7-H1 is not expressed constitutively by ECs; however, it is rapidly induced by IFN-gamma, and synergistically by IFN-gamma and TNF. In inflamed skin, B7-H1 is expressed by a subset of microvessels, and by keratinocytes, but is barely detectable in normal skin. Blocking the interaction of EC-expressed B7-H1 with its T cell ligand, programmed death-1 (PD-1), using a PD-1-Fc fusion protein, or by blocking B7-H1 expression with morpholino antisense oligonucleotides, augments expression of IL-2 and IFN-gamma, implicating B7-H1 as a negative regulator of cytokine synthesis. However, signaling through PD-1 does not affect induction of the activation markers CD25 or CD69 on T cells, suggesting that its effects are specific to cytokine synthesis. The suppressive effects of B7-H1 on cytokine expression are proportional to the strength of the primary stimulus, allowing for B7-H1 to determine the level of T cell activation in response to ECs. Our results demonstrate that B7-H1 negatively regulates cytokine synthesis in T cells activated by ECs.