Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Adenylyl cyclase inhibition and altered G protein subunit expression and ADP-ribosylation patterns in tissues and cells from Gi2 alpha-/-mice.

The inhibition of alpha i2-/- mouse cardiac isoproterenol-stimulated adenylyl cyclase (AC; EC 4.6.1.1) activity by carbachol and that of alpha i2-/- adipocyte AC by phenylisopropyladenosine (PIA), prostaglandin E2, and nicotinic acid were partially, but not completely, inhibited. While the inhibition of cardiac AC was affected in all alpha i2-/- animals tested, only 50% of the alpha i2-/- animals showed an impaired inhibition of adipocyte AC, indicative of a partial penetrance of this phenotype. In agreement with previous results, the data show that Gi2 mediates hormonal inhibition of AC and that Gi3 and/or Gi1 is capable of doing the same but with a lower efficacy. Disruption of the alpha i2 gene affected about equally the actions of all the receptors studied, indicating that none of them exhibits a striking specificity for one type of Gi over another and that receptors are likely to he selective rather than specific in their interaction with functionally homologous G proteins (e.g., Gi1, Gi2, Gi3). Western analysis of G protein subunit levels in simian virus 40-transformed primary embryonic fibroblasts from alpha i2+/+ and alpha i2-/- animals showed that alpha i2 accounts for about 50% of the immunopositive G protein alpha subunits and that loss of the alpha i2 is accompanied by a parallel reduction in G beta 35 and G beta 36 subunits and by a 30-50% increase in alpha i3. This suggests that G beta-gamma levels may be regulated passively through differential rates of turnover in their free vs. trimeric states. The existence of compensatory increase(s) in alpha i subunit expression raises the possibility that the lack of effect of a missing alpha i2 on AC inhibition in adipocytes of some alpha i2-/- animals may be the reflection of a more pronounced compensatory expression of alpha i3 and/or alpha i1.

Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy.

Like human gliomas, the rat 9L gliosarcoma secretes the immunosuppressive transforming growth factor beta (TGF-beta). Using the 9L model, we tested our hypothesis that genetic modification of glioma cells to block TGF-beta expression may enhance their immunogenicity and make them more suitable for active tumor immunotherapy. Subcutaneous immunizations of tumor-bearing animals with 9L cells genetically modified to inhibit TGF-beta expression with an antisense plasmid vector resulted in a significantly higher number of animals surviving for 12 weeks (11/11, 100%) compared to immunizations with control vector-modified 9L cells (2/15, 13%) or 9L cells transduced with an interleukin 2 retroviral vector (3/10, 30%) (P < 0.001 for both comparisons). Histologic evaluation of implantation sites 12 weeks after treatment revealed no evidence of residual tumor. In vitro tumor cytotoxicity assays with lymph node effector cells revealed a 3- to 4-fold increase in lytic activity for the animals immunized with TGF-beta antisense-modified tumor cells compared to immunizations with control vector or interleukin 2 gene-modified tumor cells. These results indicate that inhibition of TGF-beta expression significantly enhances tumor-cell immunogenicity and supports future clinical evaluation of TGF-beta antisense gene therapy for TGF-beta-expressing tumors.

cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta-adrenergic agonists.

The role of cAMP subcellular compartmentation in the progress of beta-adrenergic stimulation of cardiac L-type calcium current (ICa) was investigated by using a method based on the use of whole-cell patch-clamp recording and a double capillary for extracellular microperfusion. Frog ventricular cells were sealed at both ends to two patch-clamp pipettes and positioned approximately halfway between the mouths of two capillaries that were separated by a 5-micron thin wall. ICa could be inhibited in one half or the other by omitting Ca2+ from one solution or the other. Exposing half of the cell to a saturating concentration of isoprenaline (ISO, 1 microM) produced a nonmaximal increase in ICa (347 +/- 70%; n = 4) since a subsequent application of ISO to the other part induced an additional effect of nearly similar amplitude to reach a 673 +/- 130% increase. However, half-cell exposure to forskolin (FSK, 30 microM) induced a maximal stimulation of ICa (561 +/- 55%; n = 4). This effect was not the result of adenylyl cyclase activation due to FSK diffusion in the nonexposed part of the cell. To determine the distant effects of ISO and FSK on ICa, the drugs were applied in a zero-Ca solution. Adding Ca2+ to the drug-containing solutions allowed us to record the local effect of the drugs. Dose-response curves for the local and distant effects of ISO and FSK on ICa were used as an index of cAMP concentration changes near the sarcolemma. We found that ISO induced a 40-fold, but FSK induced only a 4-fold, higher cAMP concentration close to the Ca2+ channels, in the part of the cell exposed to the drugs, than it did in the rest of the cell. cAMP compartmentation was greatly reduced after inhibition of phosphodiesterase activity with 3-isobutyl-methylxanthine, suggesting the colocalization of enzymes involved in the cAMP cascade. We conclude that beta-adrenergic receptors are functionally coupled to nearby Ca2+ channels via local elevations of cAMP.

Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide.

The beta-amyloid peptide, the hallmark of Alzheimer disease, forms fibrillar toxic aggregates in brain tissue that can be dissolved only by strong denaturing agents. To study beta-amyloid formation and its inhibition, we prepared immune complexes with two monoclonal antibodies (mAbs), AMY-33 and 6F/3D, raised against beta-amyloid fragments spanning amino acid residues 1-28 and 8-17 of the beta-amyloid peptide chain, respectively. In vitro aggregation of beta-amyloid peptide was induced by incubation for 3 h at 37 degrees C and monitored by ELISA, negative staining electron microscopy, and fluorimetric studies. We found that the mAs prevent the aggregation of beta-amyloid peptide and that the inhibitory effect appears to be related to the localization of the antibody-binding sites and the nature of the aggregating agents. Preparation of mAbs against "aggregating epitopes," defined as sequences related to the sites where protein aggregation is initiated, may lead to the understanding and prevention of protein aggregation. The results of this study may provide a foundation for using mAbs in vivo to prevent the beta-amyloid peptide aggregation that is associated with Alzheimer disease.

d-alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.

d-alpha-Tocopherol, but not d-beta-tocopherol, negatively regulates proliferation of vascular smooth muscle cells at physiological concentrations. d-alpha-Tocopherol inhibits protein kinase C (PKC) activity, whereas d-beta-tocopherol is ineffective. Furthermore d-beta-tocopherol prevents the inhibition of cell growth and of PKC activity caused by d-alpha-tocopherol. The negative regulation by d-alpha-tocopherol of PKC activity appears to be the cause and not the effect of smooth muscle cell growth inhibition. d-alpha-Tocopherol does not act by binding to PKC directly but presumably by preventing PKC activation. It is concluded that, in vascular smooth muscle cells, d-alpha-tocopherol acts specifically through a nonantioxidant mechanism and exerts a negative control on a signal transduction pathway regulating cell proliferation.

Thyroid hormone (T3) inhibits ciprofibrate-induced transcription of genes encoding beta-oxidation enzymes: cross talk between peroxisome proliferator and T3 signaling pathways.

Peroxisome proliferators cause rapid and coordinated transcriptional activation of genes encoding peroxisomal beta-oxidation system enzymes by activating peroxisome proliferator-activated receptor (PPAR) isoform(s). Since the thyroid hormone (T3; 3,3',5-triiodothyronine) receptor (TR), another member of the nuclear hormone receptor superfamily, regulates a subset of fatty acid metabolism genes shared with PPAR, we examined the possibility of interplay between peroxisome proliferator and T3 signaling pathways. T3 inhibited ciprofibrate-induced luciferase activity as well as the endogenous peroxisomal beta-oxidation enzymes in transgenic mice carrying a 3.2-kb 5'-flanking region of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene fused to the coding region of luciferase. Transfection assays in hepatoma H4-II-E-C3 and CV-1 cells indicated that this inhibition is mediated by TR in a ligand-dependent fashion. Gel shift assays revealed that modulation of PPAR action by TR occurs through titration of limiting amounts of retinoid X receptor (RXR) required for PPAR activation. Increasing amounts of RXR partially reversed the inhibition in a reciprocal manner; PPAR also inhibited TR activation. Results with heterodimerization-deficient TR and PPAR mutants further confirmed that interaction between PPAR and TR signaling systems is indirect. These results suggest that a convergence of the peroxisome proliferator and T3 signaling pathways occurs through their common interaction with the heterodimeric partner RXR.

Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts.

The RII beta regulatory subunit of cAMP-dependent protein kinase (PKA) contains an autophosphorylation site and a nuclear location signal, KKRK. We approached the structure-function analysis of RII beta by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RII beta was replaced with Ala (RII beta-P) or Arg264 of KKRK was replaced with Met (RII beta-K). ras-transformed NIH 3T3 (DT) cells were transfected with expression vectors for RII beta, RII beta-P, and RII beta-K, and the effects on PKA isozyme distribution and transformation properties were analyzed. DT cells contained PKA-I and PKA-II isozymes in a 1:2 ratio. Over-expression of wild-type or mutant RII beta resulted in an increase in PKA-II and the elimination of PKA-I. Only wild-type RII beta cells demonstrated inhibition of both anchorage-dependent and -independent growth and phenotypic change. The growth inhibitory effect of RII beta overexpression was not due to suppression of ras expression but was correlated with nuclear accumulation of RII beta. DT cells demonstrated growth inhibition and phenotypic change upon treatment with 8-Cl-cAMP. RII beta-P or RII beta-K cells failed to respond to 8-Cl-cAMP. These data suggest that autophosphorylation and nuclear location signal sequences are integral parts of the growth regulatory mechanism of RII beta.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

cAMP-dependent, long-lasting inhibition of a K+ current in mammalian neurons.

We report the long-term modulation of K+ channels by cAMP in cultured murine colliculi neurons. A short (1-2 s) application of 8-Br-cAMP induced a long-lasting broadening of the action potential, a loss of after-hyperpolarization, and a reduction in spike accommodation. In agreement with these changes, 8-Br-cAMP produced a long-lasting (2 hr) inhibition of a K+ current. These effects were also observed after a short activation of the pituitary adenylyl cyclase-activating polypeptide, beta-adrenergic, and 5-hydroxytryptamine type 4 (5-HT4) receptors, all known to increase cAMP. A transient activation of the cAMP-dependent protein kinase and a long-lasting inhibition of phosphatases (up to 2 hr) were detected. The blockade of the K+ current resulting from a brief application of 8-Br-cAMP or 5-hydroxytryptamine was prolonged from 2 to 4 hr when protein-serine/threonine phosphatases 1 and 2A were inhibited with 10 nM okadaic acid. The critical steps following the cAMP-dependent protein kinase activation and resulting in a long-term blockade of phosphatases are discussed in this report.

Macrophage-colony-stimulating factor regulates expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 by murine bone marrow macrophages.

We observed that when monocyte/macrophage precursors derived from murine bone marrow were treated with macrophage-colony-stimulating factor (M-CSF), there was a dose-dependent increase in both the number of adherent cells and the degree to which the cells were highly spread. Attachment was supported by fibronectin, but not by vitronectin or laminin, suggesting that the integrins alpha 4 beta 1 and/or alpha 5 beta 1 might mediate this event. Binding to fibronectin was blocked partially by antibodies to either integrin, and inhibition was almost complete when the antibodies were used in combination. By a combination of surface labeling with 125I and metabolic labeling with [35S]methionine and [35S]cysteine, we demonstrated that M-CSF treatment led to increased synthesis and surface expression of the two beta 1 integrins. Since attachment to fibronectin and/or stromal cells plays an important role in the maturation of other hematopoietic lineages, we propose that the action of M-CSF in the differentiation of immature monocytes/macrophages includes stimulated expression of the integrins alpha 4 beta 1 and alpha 5 beta 1, leading to interactions with components of the marrow microenvironment necessary for cell maturation.

Overexpression of the c-Myc oncoprotein blocks the growth-inhibitory response but is required for the mitogenic effects of transforming growth factor beta 1.

One of the more intriguing aspects of transforming growth factor beta 1 (TGF beta 1) is its ability to function as both a mitogenic factor for certain mesenchymal cells and a potent growth inhibitor of lymphoid, endothelial, and epithelial cells. Data are presented indicating that c-myc may play a pivotal role in both the mitogenic and antiproliferative actions of TGF beta 1. In agreement with previous studies using C3H/10T1/2 fibroblasts constitutively expressing an exogenous c-myc cDNA, we show that AKR-2B fibroblasts expressing a chimeric estrogen-inducible form of c-myc (mycER) are able to form colonies in soft agar in the presence of TGF beta 1 only when c-myc is activated by hormone. Whereas these findings support a synergistic role for c-myc in mitogenic responses to TGF beta 1, we also find that c-myc can antagonize the growth-inhibitory response to TGF beta 1. Mouse keratinocytes (BALB/MK), which are normally growth-arrested by TGF beta 1, are rendered insensitive to the growth-inhibitory effects of TGF beta 1 upon mycER activation. This ability of mycER activation to block TGF beta 1-induced growth arrest was found to occur only when the fusion protein was induced with hormone in the early part of G1. Addition of estradiol late in G1 had no suppressive effect on TGF beta 1-induced growth inhibition.

Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene.

Conditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic beta cells in transgenic mice. A fusion protein containing the tet repressor (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the repressor into a transcription activator, was produced in beta cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in beta cells activated TAg transcription, resulting in the development of beta-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable beta-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited beta-cell proliferation. These findings indicate that transformed beta cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of beta-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest.

Overexpressed Ca-v beta 3 inhibits N-type (Ca(v)2.2) calcium channel currents through a hyperpolarizing shift of "ultra-slow" and "closed-state" inactivation

It has been shown that P auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a hovel inhibitory effect of beta3 Subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/ cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at physiological holding potentials (HPs) of -60 and -80 mV At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca,.2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced Current suppression was reversed at a HP of - 120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba-2divided by (40 mM) as a charge carrier also largely diminished the effect of P3 at -80 mV Therefore, experimental conditions (HP, divalent cation concentration, and P3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel P3 effect. Steady-state inactivation curves revealed that N-type channels exhibited closed-state inactivation without P3, and that P3 caused an similar to40 mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of P3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV Similar ultra-slow inactivation was observed for N-type channels Without P3. Thus, P3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by Causing a hyperpolarizing shift of the inactivation without affecting ultra-slow and closed-state inactivation properties.