CHO cells

Purpose. Receptors for vitamin folic acid are frequently overexpressed on epithelial cancer cells, especially ovarian cancer cells. In this study, we examined whether this expression might be exploited to specifically deliver antisense oligodeoxynucleotides (ODN) to tumor cells.

The nucleotide-sugar-activated P2Y 14 receptor (P2Y 14 -R) is highly expressed in hematopoietic cells. Although the physiologic functions of this receptor remain undefined, it has been strongly implicated recently in immune and inflammatory responses. Lack of availability of receptor-selective high-affinity antagonists has impeded progress in studies of this and most of the eight nucleotide-activated P2Y receptors. A series of molecules recently were identified by that exhibited antagonist activity at the P2Y 14 -R. We synthesized one of these molecules, a 4,7-disubstituted 2naphthoic acid derivative (PPTN), and studied its pharmacological properties in detail. The concentration-effect curve of UDP-glucose for promoting inhibition of adenylyl cyclase in C6 glioma cells stably expressing the P2Y 14 -R was shifted to the right in a concentration-dependent manner by PPTN. Schild analyses revealed that PPTN-mediated inhibition followed competitive kinetics, with a K B of 434 pM observed. In contrast, 1 mM PPTN exhibited no agonist or antagonist effect at the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , or P2Y 13 receptors. UDP-glucose-promoted chemotaxis of differentiated HL-60 human promyelocytic leukemia cells was blocked by PPTN with a concentration dependence consistent with the K B determined with recombinant P2Y 14 -R. In contrast, the chemotactic response evoked by the chemoattractant peptide fMetLeuPhe was unaffected by PPTN. UDP-glucose-promoted chemotaxis of freshly isolated human neutrophils also was blocked by PPTN. In summary, this work establishes PPTN as a highly selective high-affinity antagonist of the P2Y 14 -R that is useful for interrogating the action of this receptor in physiologic systems.

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8,...

Quantitative immunoelectron microscopy and subcellular fractionation established the site of endoplasmic reticulum (ER)-Golgi transport arrest induced by the phosphatase inhibitor okadaic acid (OA). OA induced the disappearance of transitional element tubules and accumulation of the anterograde-transported Chandipura (CHP) virus G protein only in the rough ER (RER) and not at more distal sites. The block was specific to the early part of the anterograde pathway, because CHP virus G protein that accumulated in the intermediate compartment (IC) at 15°C could gain access to Golgi stack enzymes. OA also induced RER accumulation of the IC protein p53/p58 via an IC-RER recycling pathway which was resistant to OA and inhibited by the G protein activator aluminium fluoride. The role of COPII coats in OA transport block was investigated by using immunofluorescence and cell fractionation. In untreated cells the COPII coat protein sec 13p colocalized with p53/p58 in Golgi-IC structures of the ...

Many persistent pesticides have been implicated in reproductive and developmental adverse effects, in man and wildlife. It has been hypothesized that these so-called xeno-hormones could upset the endocrine system function by binding to human estrogen receptor alpha and beta (ERα, β) and thus be responsible for the higher incidence of breast and cervical cancer, infertility and endometriosis. In this report, forty-nine pesticides were tested for ERα and β activation or inhibition in stable reporter cell lines, HELN ERα and ERβ. Stable transfection of the ERα and ERβ constructs together with an estrogen reporter luciferase vector into the HeLa cell line resulted in two estradiol-sensitive cell lines. In our model, fifteen of the tested pesticides were found to agonize the ERα-mediated transcription in a dose-dependent manner and DDT, transnonachlor, chlordane, fenvalerate and toxaphene were also capable to activate ERβ. Antagonistic activities toward hERα and hERβ were shown in three (carbaryl, pentachlorophenol and 2,4,5-trichlorophenoxyacetic acid) and seven (chlordecone, methoxychlor, carbaryl, endosulfan, endrin, dieldrin, aldrin) pesticides, respectively. Remarkably chlordecone and methoxychlor which were the most effective antagonist compounds for hERβ, were agonists for hERα. Although the ERα activation potential of the pesticides was lower than that of estradiol, the overall body scale response might be amplified by the ability of pesticides to act via several mechanisms and by frequent and prolonged exposure to different pesticides, even at low concentrations.

The behavior of chimeric proteins consisting of A-type lamins and green fluorescent protein (GFP) was studied to investigate the localization and dynamics of nuclear lamins in living cells. Cell line CHO-K1 was transfected with cDNA constructs encoding fusion proteins of lamin A-GFP, lamin AΔ10-GFP, or lamin C-GFP. In the interphase nucleus lamin-GFP fluorescence showed a perinuclear localization and incorporation into the lamina for all three constructs. Our findings show for the first time that the newly discovered lamin AΔ10 protein is localized to the nuclear membrane. The GFP-tagged lamins were processed and behaved similarly to the endogenous lamin molecules, at least in cells that expressed physiological levels of the GFP-lamins. In addition to the typical perinuclear localization, in the majority of transfected cells each individual A-type lamin-GFP revealed an extensive collection of branching intra- and trans-nuclear tubular structures, which showed a clear preference for ...

Here, we combined classical biochemistry with novel biophysical approaches to study with high spatial and temporal resolution the organization of GPI-anchored proteins (GPI-APs) at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, following sorting in the Golgi, each GPI-AP reaches the apical surface in homo-clusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesteroldependent hetero-clusters. By contrast, in non-polarized MDCK cells GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form hetero-clusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, different from fibroblasts, in polarized epithelial cells a selective cholesteroldependent sorting mechanism in the Golgi regulates both the organization and the function of GPI-APs at the apical surface.

We explored the interaction of a nitrogen mustard derivative of acetylcholine with the human M 2 muscarinic receptor expressed in CHO cells using the muscarinic radioligand, [ 3 H]Nmethylscopolamine. Acetylcholine mustard caused a concentration-dependent, first order loss of [ 3 H]N-methylscopolamine binding at 37°C, with the half maximal rate constant occurring at 24 µM and a maximal rate constant of 0.16 min -1 . We examined the effects of various ligands on the rate of alkylation of M 2 receptors by acetylcholine mustard. N-methylscopolamine and McN-A-343 (4-(trimethylamino)-2-butynyl-(3-chlorophenyl)carbamate) competitively slowed the rate of alkylation, whereas the inhibition by gallamine reached a plateau at high concentrations, indicating allosteric inhibition. In contrast, WIN 51708 (17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2-b] pyrimido[1,2-a]benzimidazole) had no effect. We also measured the inhibition of [ 3 H]NMS binding by acetylcholine mustard at 0°C, conditions under which there is little or no detectable covalent binding. In these experiments, the dissociation constant of the aziridinium ion of acetylcholine mustard was estimated to be 12.3 µM. In contrast, the parent mustard and alcoholic hydrolysis product of acetylcholine mustard were without effect. Our results show that measurement of the effects of ligands on the rate of inactivation of the orthosteric site by a small site-directed electrophile is a powerful method for discriminating competitive inhibition from allosterism.

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The effect of supplementation with the omega 3 polyunsaturated fatty acid (n-3 PUFA) docosahexaenoic acid (DHA) on membrane composition and amyloid-β1-42 (Aβ42) secretion was studied in human amyloid-β protein precursor-transfected Chinese Hamster Ovary (CHO) cells. Twenty-four hour incubation with a range of DHA concentrations resulted in a dosedependent increase in membrane DHA and eicosapentaenoic acid content and a decrease in arachidonic acid content. In addition, DHA supplementation caused a dose-dependent reduction in the secreted Aβ42 levels and resulted in a 4-8 fold decrease in extracellular prostaglandin E2 (PGE2) levels. Tocopherol, which was added to DHA to prevent oxidation, may have contributed to the effect of DHA, since it slightly decreased extracellular Aβ42 and PGE2 levels when given alone. The addition of selective COX-2 inhibitors Celebrex and curcumin to the culture medium resulted in a significant and comparable inhibition of PGE2 release, but did not inhibit Aβ42 secretion, and even significantly increased Aβ42 production in this cell system. Together, the present data show that, whereas both DHA and COX-2 inhibitors may reduce PGE2 production, only DHA in the presence of tocopherol significantly reduced Aβ42 production and concurrently changed membrane lipid composition in CHO cells. It is concluded that in this in vitro setting DHA reduced Aβ42 secretion through membrane-related, but not PGE2-related mechanisms.

CD4-immunoglobulin G2 (IgG2) is a fusion protein comprising human IgG2 in which the Fv portions of both heavy and light chains have been replaced by the V1 and V2 domains of human CD4. Previous studies found that CD4-IgG2 potently neutralizes a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates in vitro and ex vivo. The current report demonstrates that CD4-IgG2 protects against infection by primary isolates of HIV-1 in vivo, using the hu-PBL-SCID mouse model. Passive administration of 10 mg of CD4-IgG2 per kg of body weight protected all animals against subsequent challenge with 10 mouse infectious doses of the laboratory-adapted T-cell-tropic isolate HIV-1 LAI , while 50 mg of CD4-IgG2 per kg protected four of five mice against the primary isolates HIV-1 JR-CSF and HIV-1 AD6 . In contrast, a polyclonal HIV-1 Ig fraction exhibited partial protection against HIV-1 LAI at 150 mg/kg but no significant protection against the primary HIV-1 isolates. The results d...

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor ␣IIb␤3 in a patient with an ill defined platelet disorder: one in the ␤3 gene (S527F) and two in the ␣IIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant ␣IIb␤3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the ␤3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated ␣IIb␤3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal antiligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of ␣IIb␤3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the ␣IIb␤3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe 527 in ␤3 and the calf-1 domain in ␣IIb and by decreased flexibility between I-EGF2 and I-EGF3.

Recent evidences suggest that Ab peptides modulate endothelial cell (EC) functions. At low concentrations, Ab 1-40 enhances the pro-angiogenic activity of FGF-2, whereas deposition of excess Ab causes EC dysfunction and cerebral amyloid angiopathy (CAA). We investigated whether FGF-2 attenuates EC dysfunction caused by pathological Ab levels. We studied Ab 1-40 on EC survival, as well as on signals responsible of their angiogenic phenotype. At 5-50 lM Ab 1-40 reduced EC population, caused apoptosis, downregulated FGF-2 production, inhibited FGF-2 binding to heparin, and FGFR1 phosphorylation. Toxic effects were owing to lack of FGF-2 stimulation, as EC overexpressing FGF-2 displayed extraordinary resistance to Ab 1-40 injuries. The FGF-2 mechanism responsible for reversing damages, involves the downstream enhancement of Akt, a pathway independent of eNOS activation. In conclusion, we demonstrate that FGF-2 protects EC from the effects of excess Ab 1-40 , suggesting that it may attenuate the consequences of Ab deposition in pathologies as CAA.

measurements provided further evidence that the disulfide-bonded enzyme retains structure in the pres-Chinese hamster ovary cells were stably transence of denaturants. Taken together, these results fected with the cDNA for human carbonic anhydrase show that the disulfide bonds contribute significantly IV that was engineered to encode a secretory form of both to the retention of structure and of catalytic acthe normally glycosylphosphatidylinositol-anchored tivity in the presence of denaturants, and to the ability membrane protein. Overexpression was achieved by to renature following removal of denaturants. ᭧ 1996 amplification of the cDNA and its dihydrofolate re-Academic Press, Inc. ductase-containing expression vector by growth in the presence of methotrexate. The 33-kDa secretory form of the enzyme was purified to homogeneity from cellular secretions by inhibitor affinity chromatography. Occasional CA IV preparations contained proteo-Carbonic anhydrase IV (CA IV) 2 is expressed on the lytic fragments of 18 and 15 kDa held together by diplasma membrane of epithelial and endothelial cells sulfide bonds. N-terminal sequencing identified the of microcapillaries of several organs and anchored to 18-kDa fragment as the N-terminus and the 15-kDa membranes by a glycosylphosphatidylinositol (GPI) anfragment as the C-terminal portion. The specific acchor (1-13). It plays a key role in reabsorption of tivity of the purified enzyme preparations (2587 { 149 HCO 0 3 in kidney (6). It is also implicated in homeostasis U/mg protein) was comparable to that of enzyme puriof CO 2 and HCO 0 3 in brain (10), in catalysis of CO 2 fied from human tissues. exchange and local pH regulation in lung (9), in acidifi-In order to identify the cysteines involved in the two cation of epididymal fluid in reproductive organs (8), disulfide bonds, enzyme purified following metabolic and in catalysis of the hydration of metabolically prolabeling with [ 35 S]cysteine was subjected to proteolytic duced CO 2 in muscle during exercise (12). cleavage and the N-terminal amino acid sequence de-The remarkable resistance of CA IV to SDS concentermined on the labeled peptides isolated by HPLC. trations that inactivate other CAs, first noted by Whit-Results indicated that the disulfide bonds in the native ney and Briggle (1), greatly facilitates its purification enzyme link Cys6 to Cys18 and Cys28 to Cys211. from tissues containing other CA isoforms. CA IV has Reduction of the enzyme or reduction followed by been purified from lung and kidney membranes from alkylation both destroy 70% of the enzyme activity and several mammalian species (1-4, 13). The human enmake the enzyme susceptible to inactivation by denazyme is unique among mammalian CA IVs in that it turants. Furthermore, the loss of activity of reduced contains no N-linked oligosaccharide chains, which are enzyme on exposure to denaturants is not recovered on removal of denaturants. By contrast, disulfide-found in variable numbers in all other CA IVs (4). The bonded enzyme is not only more resistant to inactivation by denaturants, but any loss of activity is reversed

The contribution of exogenous and endogenous TGF-beta 1 to human peripheral blood NK cell proliferation and activity in vitro was investigated. Exogenous bioactive TGF-beta 1 inhibited NK cell DNA synthesis and production of IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF) by NK cultures consisting of > 98% CD56+ cells. The cytotoxic activity of NK cells was also weakly inhibited by exogenous TGF-beta 1. All TGF-beta 1-induced inhibitory effects occurred in the absence and presence of the NK cell-activating cytokines IFN-alpha, IL-2, and IL-12. Unstimulated NK cell cultures expressed steady state TGF-beta 1 mRNA detected by Northern blot analysis and produced TGF-beta protein (1.6 ng/ml), as determined by ELISA. When NK cell proliferation was induced by IL-2, IL-12, IFN-alpha, or a combination of IL-2 and IL-12, expression of TGF-beta 1 mRNA and protein was moderately and consistently reduced by approximately 20%, as compared with unstimulated control cultures. Unsti...

MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylatedO-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating...

Monoclonal antibodies continue to command a large market for treatment of a variety of diseases. In many cases, the doses required for therapeutic efficacy are large, limiting options for antibody delivery and administration. We report a novel formulation strategy based on dispersions of antibody nanoclusters that allows for subcutaneous injection of highly concentrated antibody (~190 mg/ml). A solution of monoclonal antibody 1B7 was rapidly frozen and lyophilized using a novel spiral-wound in situ freezing technology (SWIFT) to generate amorphous particles. Upon gentle stirring, a translucent dispersion of ~430 nm protein clusters low apparent viscosity (~24 cp) formed rapidly in buffer containing the pharmaceutically acceptable crowding agents, trehalose, polyethylene glycol and n-methyl-2-pyrrolidone. Upon in vitro dilution of the dispersion, the nanoclusters rapidly reverted to monomeric protein with full activity, as monitored by dynamic light scattering and antigen binding. When administered to mice as an intravenous solution, subcutaneous solution or subcutaneous dispersion at similar (4.6-7.3 mg/kg) or ultra-high dosages (51.6 mg/kg), the distribution and elimination kinetics were within error and the protein retained full activity. Overall, this method of generating high-concentration, low-viscosity dispersions of antibody nanoclusters could lead to improved administration and patient compliance, providing new opportunities for the biotechnology industry.

In both pro-and eukaryotes, the mutagenic and toxic DNA adduct O 6 -methylguanine (O 6 MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O 6 -alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N -nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O 6 -methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O 6 -chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O 6 MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O 6 MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.

Nogo-A is a potent neurite growth inhibitorin vitroand plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-te...

The adult mammalian CNS has a limited capacity for nerve regeneration and structural plasticity. The presence of gliaderived inhibitory factors myelin-associated glycoprotein (MAG) and Nogo-A have been suggested to provide a nonpermissive environment for elongating nerve fibers. In particular, Nogo-A, an integral membrane protein predominantly expressed by oligodendrocytes, has been demonstrated to impair neurite growth in vitro and in vivo. Structure function analysis revealed that Nogo-A protein contains at least two active domains, NiG and Nogo-66, with diverse effects on neurite outgrowth and cell spreading. We now provide evidence that these inhibitory domains mediate their effects via an antagonistic regulation of the small GTPases RhoA and Rac1, resulting in activation of RhoA and suppression of Rac1. By inactivating RhoA with C3 transferase or the downstream effector Rhokinase ROCK with Y27632, the inhibitory effects of both Nogo-A fragments and MAG on neurite outgrowth and oligodendrocyte-mediated growth cone collapse were abolished. Furthermore, we show that the recently cloned receptor for Nogo-66 and MAG, NgR, is not necessary for either NiG-or MAG-induced RhoA activation.

Myelin is a major obstacle for regenerating nerve fibers of the adult mammalian central nervous system (CNS). Several proteins including Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and the chondroitin-sulfate proteoglycan (CSPG) Versican V2 have been identified as inhibitory components present in CNS myelin. MAG, OMgp as well as the Nogo specific domain Nogo-66 exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor NgR and the neurotrophin receptor p75(NTR). While this suggests a converging role of the p75(NTR)/NgR receptor complex for myelin-derived neurite growth inhibitors, we show here that NgR/p75(NTR) is not required for mediating the inhibitory activity of the two myelin components NiG, unlike Nogo-66 a distinct domain of Nogo-A, and Versican V2. Primary neurons derived from a complete null mutant of p75(NTR) are still sensitive to NiG and Versican V2. In line with this result, neu...

Nogo-A is a potent neurite outgrowth inhibitory protein in vitro and is suggested to play a role in the lack of regeneration in the central nervous system of adult higher vertebrates. A shorter splice isoform, ASY/Nogo-B, has recently been reported to act as a proapoptotic protein, the loss of which would be typical for cancer cells. Here, we show that the osteosarcoma cell line SaOS-2 and the cell line CHO do express high levels of endogenous Nogo-B and that stable transfectants overexpressing high levels of Nogo-B do not differ significantly from the respective parental wild-type or control cell lines both in respect to cell proliferation and to spontaneous apoptosis or cell death induced by staurosporine and tunicamycin. The deletion of the second transmembrane domain of Nogo-B, which has been claimed to abolish its proapoptotic activity, leads to a shift of the protein from the ER to a cytoplasmic localization, suggesting that ER stress of highly overexpressed Nogo-B may lead to aversive cellular reactions under particular conditions. Our data do not support a function of Nogo-B as a physiological proapoptotic protein in certain types of cancer.

Nogo-A is a protein associated with central nervous system (CNS) myelin thought to impair regenerative responses and to suppress sprouting and plastic changes of synaptic terminals. In this study, we report that serum IgM autoantibodies to the recombinant large Nterminal inhibitory domain of Nogo-A are a frequent finding in multiple sclerosis (MS) and acute inflammatory (IND) and non-inflammatory neurological diseases (OND), but not in neurodegenerative diseases (ND), systemic inflammatory disease and healthy controls. Furthermore, we demonstrate intrathecal production of anti-Nogo-A antibodies measured by increased IgG indices. Intrathecal anti-Nogo antibodies were significantly more frequent in patients with relapsing -remitting as compared to chronic progressive (CP) MS. We also found a highly significant negative correlation of these antibody responses with age indicating that they are more frequent in younger patients. We finally demonstrate that human anti-Nogo-A antibodies recognize native Nogo-A in brain extracts, oligodendrocytes and cells expressing human Nogo-A.

Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis. In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling. Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk. These studie...

The discovery, synthesis and structure-activity relationship (SAR) of a novel series of cannabinoid 1 (CB 1 ) and cannabinoid 2 (CB 2 ) receptor ligands are reported. Based on the aminoalkylindole class of cannabinoid receptor agonists, a biphenyl moiety was introduced as novel lipophilic indole 3-acyl substituent in 11À16. Furthermore, the 3-carbonyl tether was replaced with a carboxamide linker in 17À20 and the azaindole (pyrrolopyridine) nucleus was designed as indole bioisostere with improved physicochemical properties in 21À25. Through these SAR efforts, several high affinity CB 1 /CB 2 dual cannabinoid receptor ligands were identified. Indole-3-carboxamide 17 displayed single-digit nanomolar affinity and w80 fold selectivity for CB 1 over the CB 2 receptor. The azaindoles displayed substantially improved physicochemical properties (lipophilicity; aqueous solubility). Azaindole 21 elicited potent cannabinoid activity. Cannabinoid receptor agonists 17 and 21 potently modulated excitatory synaptic transmission in an acute rat brain slice model of cannabinoid receptor-modulated neurotransmission.

Poly(amidoamine) (PAMAM) dendrimers are promising multipotent gene delivery vectors, providing favorable DNA condensation properties also in combination with the possibility of conjugation of different targeting ligands to their surface. They have been used for transfection both in vitro and in vivo, but their application is currently somewhat limited due to inherent cytotoxicity. In this work we investigate how two types of surface modification, acetylation and PEGylation, affect the DNA binding characteristics, the cytotoxicity and the in vitro transfection efficiency of generation 4 and 5 PAMAM dendrimers. Particularly, we address how the morphology of DNA-dendrimer complexes, formed under low salt conditions, changes upon dilution in cell growth medium, an event that inevitably occurs before the complexes reach the cell surface in any transfection experiment. We find that acetylation and PEGylation essentially eliminates the inherent dendrimer cytotoxicity. However, the transfection efficiency of the modified dendrimers is lower than that of the corresponding unmodified dendrimers, which can be rationally understood by our observations that DNA is less condensed when complexed with these modified dendrimers. Although small DNA-dendrimer particles are formed, the availability for ethidium intercalation and nuclease degradation is significantly higher in the modified DNA-dendrimer complexes than in unmodified ones. Dilution in cell growth medium has a drastic effect on these electrostatically assembled complexes, resulting in increase in size and DNA availability. Our results strongly add to the notion that it is of importance to perform a biophysical characterization under conditions as close to the transfection situation as possible, to enable conclusions regarding structure-activity relations of gene delivery vectors.

The c-Met pathway is frequently deregulated in a wide variety of human malignancies and its overexpression is known to play a key role in tumor development and metastasis. Inhibiting c-Met signaling is emerging as a promising strategy and several inhibitors are at various stages of clinical development. This study describes the generation of a humanized anti-cMet antibody able to inhibit both ligand-dependent and -independent tumor growth in a sustainable fashion.

ABSTRACTThe diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamilyGammaherpesvirinaeand currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein asRCP) in infected rhesus macaque fibroblasts mapped the ends of the transcript...

Rhesus rhadinovirus (RRV) is currently the closest known, fully sequenced homolog of human Kaposi sarcoma-associated herpesvirus. Both these viruses encode complement inhibitors as follows: Kaposi sarcoma-associated herpesvirus-complement control protein (KCP) and RRV-complement control protein (RCP). Previously we characterized in detail the functional properties of KCP as a complement inhibitor. Here, we performed comparative analyses for two variants of RCP protein, encoded by RRV strains H26-95 and 17577. Both RCP variants and KCP inhibited human and rhesus complement when tested in hemolytic assays measuring all steps of activation via the classical and the alternative pathway. RCP variants from both RRV strains supported C3b and C4b degradation by factor I and decay acceleration of the classical C3 convertase, similar to KCP. Additionally, the 17577 RCP variant accelerated decay of the alternative C3 convertase, which was not seen for KCP. In contrast to KCP, RCP showed no affinity to heparin and is the first described complement inhibitor in which the binding site for C3b/C4b does not interact with heparin. Molecular modeling shows a structural disruption in the region of RCP that corresponds to the KCP-heparin-binding site. This makes RRV a superior model for future in vivo investigations of complement evasion, as RCP does not play a supportive role in viral attachment as KCP does.

The human microsomal 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) metabolizes active cortisol into cortisone and protects the mineralocorticoid receptor from glucocorticoid occupancy. In a congenital deficiency of 11β-HSD2, the protective mechanism fails and cortisol gains inappropriate access to mineralocorticoid receptor, resulting in low-renin hypertension and hypokalemia. In the present study, we describe the clinical and molecular genetic characterization of a patient with a new mutation in the HSD11B2 gene. This is a 4-yr-old male with arterial hypertension. The plasma renin activity and serum aldosterone were undetectable in the presence of a high cortisol to cortisone ratio. PCR amplification and sequence analysis of HSD11B2 gene showed the homozygous mutation in exon 4 Asp223Asn (GAC→AAC) and a single nucleotide substitution C→T in intron 3. Using site-directed mutagenesis, we generated a mutant 11βHSD2 cDNA containing the Asp223Asn mutation. Wild-type and mutant cDNA ...

Histamine H2 receptors transfected in Chinese hamster ovary (CHO) cells are time- and dose-dependently upregulated upon exposure to the H2 antagonists cimetidine and ranitidine. This effect appears to be H2 receptor-mediated as no change in receptor density was observed after H1 or H3 antagonist treatment or after incubation with the structural analogue of cimetidine, VUF 8299, which has no H2 antagonistic effects. By using transfected CHO cells expressing different densities of wild-type H2 receptors or an uncoupled H2Leu124Ala receptor, the histamine H2 receptor was found to display considerable agonist-independent H2 receptor activity. Cimetidine and ranitidine, which both induce H2 receptor upregulation, actually functioned as inverse agonists in those cell lines displaying spontaneous agonist-independent H2 receptor activity. Burimamide, on the other hand, was shown to act as a neutral antagonist and did as expected not induce H2 receptor upregulation after long-term exposure. ...

S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.

MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many autoimmune diseases. Understanding the role of HLA class II haplotypes in immunity is hampered by the lack of animal models expressing these genes as authentic cis-haplotypes. In this study we describe transgenic expression of the autoimmune prone HLA DR3-DQ2 haplotype from a yeast artificial chromosome (YAC) containing an intact ∼320-kb region from HLA DRA to DQB2. In YAC-transgenic mice HLA DR and DQ gene products were expressed on B cells, macrophages, and dendritic cells, but not on T cells indicating cell-specific regulation. Positive selection of the CD4 compartment by human class II molecules was 67% efficient in YAC-homozygous mice lacking endogenous class II molecules (Aβnull/null) and expressing only murine CD4. A broad range of TCR Vβ families was used in the peripheral T cell repertoire, which was also purged of Vβ5-, Vβ11-, and Vβ12-bearing T cells by endogenous mouse mamma...

The mechanisms of activation of cytoplasmic phospholipase A, (cPLA,) are complex and incompletely defined. In Chinese hamster ovary (CHO) cells, receptor stimulation of cPLA, is due to the interaction of pathways involving the a subunits of at least two guanine-nucleotide-binding (G) proteins, Ga,, and Ga,. Activation of cPLA, is inhibited by pertussis toxin and Gai, mutants. In addition, activation of phospholipase C via Ga, results in increased intracellular calcium ([Ca'+],) and activation of protein kinase C, both of which interact with and activate cPLA,. The present study was undertaken to analyze the mechanism of interaction of Gai, with the phospholipase-C-stimulated pathway in the activation of cPLA,. We addressed this question using a dominant negative Ga,, mutant, [G203T]Ga,,, in which Gly203 is mutated to Thr. [G203T]Gai, inhibits ATP receptor activation of cPLA,. The effect of [G203T]Ga,, was specific to Gag,-activated pathways, as shown by its lack of effect on other purinergic receptor stimulated pathways : ATP stimulation of [Ca2+Ii or mitogen-activated protein kinase phosphorylation is unaltered by [G203T]Ga,,. We addressed the possibility that the activation of cPLA, by Ca2+ and/or protein kinase C is dependent on Ga,,. Activation of cPLA, by the Ca" ionophore, ionomycin, was inhibited by 61 ? 9% (n = 5) in [G203T]Gai,-expressing cells; however the ionomycin-induced [Ca"], rise was unaffected by [G203T]Gai,. Thus, [G203T]Gai, specifically inhibits Ca2+ activation of cPLA,. In contrast, activation of cPLA, via protein kinase C by phorbol 12-myristate 13-acetate was unaffected by [G203T]Gai,. Our results demonstrate that Ca" but not phorbol ester activation of cPLA, in CHO cells is Ga,-dependent. The possibility is discussed that Ga,, is downstream of Ca2+ but upstream of protein kinase C activation of cPLA,.

The SDYQRL motif of the cytoplasmic domain of TGN38 is involved in targeting TGN38 from endosomes to the TGN. To create a system for studying this pathway, we replaced the native transferrin receptor (TR) internalization motif (YTRF) with the SDYQRL TGN-targeting motif. The advantages of using TR as a reporter molecule include the ability to monitor trafficking, in both biochemical and microscopy experiments, using the natural ligand transferrin. When expressed in CHO cells, the SDYQRL-TR construct accumulated in juxtanuclear tubules and vesicles that are in the vicinity of the TGN. The SDYQRL-TR-containing structures, however, do not colocalize with TGN markers (e.g., NBD ceramide), and therefore the SDYQRL motif is not sufficient to target the TR to the TGN. The morphology of the SDYQRL-TR-containing juxtanuclear structures is different from the recycling compartment found in cells expressing the wild-type TR. In addition, the SDYQRL-TR-containing juxtanuclear compartment is more ...

SummaryLittle is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins. These adhesins recognize as receptors the GPI‐anchored proteins CD55 (decay‐accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA). CD66e is a member of the CEA‐related cell adhesion molecules (CEACAM) family, comprising seven members. We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM‐expressing CHO and HeLa cells. The results demonstrate that only E. coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr‐I, including Dr, F1845 and AfaE‐III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6. Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr‐I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6. In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recr...

We used transfected epithelial CHO-B2 cells as a model to identify the mechanism mediating internalization of Afa/Dr diffusely adhering Escherichia coli . We provide evidence that neither the α5 or β1 integrin subunits nor α5β1 integrin functioned as a receptor mediating the adhesion and/or internalization of Dr or Afa-III fimbria-positive bacteria. We also demonstrated that (i) whether or not the AfaD or DraD invasin subunits were present, there was no difference in the cell association and entry of bacteria and that (ii) DraE or AfaE-III adhesin subunits are necessary and sufficient to promote the receptor-mediated bacterial internalization into epithelial cells expressing human decay-accelerating factor (DAF), CEACAM1, CEA, or CEACAM6. Internalization of Dr fimbria-positive E. coli within CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells occurs through a microfilament-independent, microtubule-dependent, and lipid raft-dependent mechanism. Wild-type Dr fimbria-positive bacteria ...

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Localized cytokine therapies with recombinant monoclonal antibody-cytokine fusion proteins, designated immunocytokines, have become of increasing interest for tumor immunotherapy, since they direct immunomodulatory cytokines into the tumor microenvironment. To investigate their mechanisms of action in a variety of syngeneic tumor models, recombinant mouse cytokines IL2 and GM-CSF were engineered as fusion proteins to the carboxyl terminus of a chimeric rat/mouse antitransferrin receptor antibody, ch17217 and expressed in stable-transfected Chinese hamster ovary cells. The recombinant immunocytokines were readily purified by affinity chromatography and their binding characteristics were identical to those shown for the ch17217 antibody. The IL2 immunocytokine had an activity similar to recombinant mouse IL2, whereas the GM-CSF immunocytokine had enhanced cytokine activity relative to recombinant mouse GM-CSF. The clearance rates of ch17217 and the GM-CSF and IL2 immunocytokines were relatively similar with elimination phases (t 1/2R ) of 1.8 h and distribution phases (t 1/2β ) of 83, 88, and 91 h, respectively. Both immunocytokines demonstrated effective antitumor activity by suppressing the growth of hepatic metastases of mouse neuroblastoma and pulmonary metastases of mouse colon carcinoma in syngeneic A/J and BALB/c mice, respectively. These results indicate that biologically effective IL2 and GM-CSF immunocytokines combine the targeting ability of an antitransferrin receptor monoclonal antibody with the immunomodulatory functions of each cytokine. Because of the universal expression of the transferrin receptor on mouse tumor cell lines, these constructs should prove useful to determine their efficacy in a wide variety of syngeneic mouse tumor models and to perform detailed studies of their modes of action.

In Shaker-group potassium channels the presence of a tyrosine residue, just downstream of the pore signature sequence GYG, determines sensitivity to tetraethylammonium (TEA). The KCNQ family of channels has a variety of amino acid residues in the equivalent position. We studied the eect of TEA on currents generated by KCNQ homomers and heteromers expressed in CHO cells. We used wild-type KCNQ1 ± 4 channels and heteromeric KCNQ2/3 channels incorporating either wild-type KCNQ3 subunits or a mutated KCNQ3 in which tyrosine replaced threonine at position 323 (mutant T323Y). IC 50 values were (mM): KCNQ1, 5.0; KCNQ2, 0.3; KCNQ3, 430; KCNQ4, 3.0; KCNQ2+KCNQ3, 3.8; and KCNQ2+KCNQ3(T323Y), 0.5. While the high TEA sensitivity of KCNQ2 may be conferred by a tyrosine residue lacking in the other channels, the intermediate TEA sensitivity of KCNQ1 and KCNQ4 implies that other residues are also important in determining TEA block of the KCNQ channels.

To investigate the fate of unrejoined DNA double-strand breaks, the frequency of 6°Co y-ray-and restriction-enzyme-induced terminal chromosome deletions, a marker of unrejoined breaks, was determined in CHO-K1 and in xrs-5 cells. The xrs-5 cell is a DNA double-strand break repair-deficient derivative of CHO-K1. Terminal deletion frequency was small in both CHO-K1 and xrs-5 cells when cells were irradiated or treated with restriction enzyme while in the G 1 phase of the cell cycle. In contrast, previous studies have shown that treatment of cells in G 2 leads to large deletion frequencies, especially in xrs-5 cells. Cell cycle analyses show large G 2 blocks in irradiated xrs-5 cells with only partial recovery over a 24-96-h period. These results suggest that most CHO cells with unrejoined breaks are blocked in G 2 and, therefore, do not contribute to chromosome mutation frequencies. The small frequencies of terminal deletions that are found in these cells may reflect either an inefficiency in the G 2 checkpoint mechanism or, perhaps, a modification of broken ends that allows passage through G 2.

We report a case of ulcer bed infection in an enlarging venous leg ulcer without clinical signs of cellulitis in the surrounding tissues. Signs of infection in the leg ulcer were: 1) cocci-like structures and bacteria-like rods around vessel walls in the viable ulcer bed, 2) vasculitis-like inflammation of deeply situated vessels of the viable tissue, 3) Pseudomonas aeruginosa-specific antibodies in the serum (other than against exotoxin A), 4) extensive epidermolysis of normal human skin by the wound exudate in vitro, and 5) P. aeruginosa exotoxin A in the wound exudate (23 ng/ml). In an in vitro cell assay, the wound exudate was cytotoxic and rabbit antibodies to exotoxin A, but not a serine proteinase inhibitor, inhibited this cytotoxicity. P. aeruginosa exotoxin A might contribute to the pathogenesis of the ulcer enlargement. The ulcer improved after the third skin graft, probably mainly due to effective treatment with a long-stretch compression bandage.

It has been postulated that intracellular binding sites for platelet-activating factor (PAF) contribute to proinflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCECs) produced PAF-molecular species in response to H2O2. Using FACS analysis, we demonstrated the expression of PAF receptors on cell and nuclear surfaces of PCECs. Confocal microscopy studies performed on PCECs, Chinese hamster ovary cells stably overexpressing PAF receptors, and isolated nuclei from PCECs also showed a robust nuclear distribution of PAF receptors. Presence of PAF receptors at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with methylcarbamate-PAF evoked a decrease in cAMP production and a pertussis toxin-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a pertussis toxin-in...