Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP)

The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.     

Ketopyrrolidines and ketoazetidines as potent dipeptidyl peptidase IV (DPP IV) inhibitors

In this paper, the synthesis and structure-activity relationships (SAR) of two classes of electrophile-based dipeptidyl peptidase IV (DPP IV) inhibitors, the ketopyrrolidines and ketoazetidines, is discussed. The SAR of these series demonstrate that the 2-thiazole, 2-benzothiazole, and 2-pyridylketones are optimal S1' binding groups for potency against DPP IV. In addition, both cyclohexyl glycine (CHG) and octahydroindole carboxylate (OIC) serve as the most potent S2 binding groups within each series. Stereochemistry at the alpha-position of the central ring is relevant to potency within the ketopyrrolidines series, but not in the ketoazetidine series. Finally, the ketoazetidines display enhanced stability over the corresponding ketopyrrolidines, while maintaining their potency. In fact, certain stabilized ketoazetidines can maintain their in vitro potency and inhibit DPP IV in the plasma for up to 6h.     

Immunotherapy with autologous tumor cells engineered to secrete Tag7/PGRP, an innate immunity recognition molecule

BACKGROUND: Recent studies indicate that the innate component of immune defense plays an important role in the establishment of antigen-specific immune response. We have previously isolated a novel mouse gene tag7/PGRP that was shown to be involved in the innate component of the immune system, and its insect homologue is an upstream mediator of Toll signaling in Drosophila. METHODS: Transiently or stably genetically modified mouse tumor cell lines expressing Tag7 were used. Tumor growth rate and animal survival were analyzed. Possible effector cells involved in tumor suppression were detected immunohistochemically. RESULTS: Transfection of mammary gland adenocarcinoma cells with the tag7 cDNA did not alter their growth rate in vitro but diminished their tumorogenicity in vivo in syngeneic and immunodeficient animals. Increased incidence of apoptosis was registered in the modified tumors. Transient expression of Tag7 by mouse melanoma M3 cells elicited protective immunity against parental tumor cells. Immunohistochemical analysis revealed that tumors after immunization with the genetically modified cells were infiltrated with Mac1(+) cells, B220(+) cells, and NK cells. Using nude mice we observed rejection of modified cells, but did not detect memory formation. CONCLUSIONS: We can conclude that secretion of the Tag7 protein by genetically modified cells can induce mobilization of antigen-presenting cells and innate effectors. Memory mechanisms are mediated by T cell response. For the first time our results demonstrate that local secretion of Tag7-the molecule involved in innate immunity-may play an important role in the induction of effective antitumor response in mice.     

Effect of Low pH on Glutamate Uptake and Release in Isolated Presynaptic Endings from Rat Brain

The effect of acidification of the incubation medium on the membrane potential and glutamate uptake and release was studied in isolated presynaptic neuronal endings (synaptosomes) from rat brain. Using the fluorescent probe diS-C₃-(5), a rapid depolarization of plasma membrane was detected at pH 6.0, most probably as a result of the inhibition of the sodium pump and potassium channel blockade. The membrane potential decrease did not result in increase of basal efflux of glutamate. Glutamate release following K⁺-induced depolarization was decreased upon lowering pH to 6.0. Acidosis inhibited mainly calcium-dependent (vesicular) release of glutamate and did not significantly reduce [¹⁴C]glutamate uptake. This inhibition of glutamate release but not of glutamate uptake may be a mechanism of the protective effect of acidosis during brain ischemia.     

Ablation of eosinophils leads to a reduction of allergen-induced pulmonary pathology

A strategy to deplete eosinophils from the lungs of ovalbumin (OVA)-sensitized/challenged mice was developed using antibody-mediated depletion. Concurrent administration [viz. the peritoneal cavity (systemic) and as an aerosol to the lung (local)] of a rat anti-mouse CCR3 monoclonal antibody resulted in the abolition of eosinophils from the lung such that the airway lumen was essentially devoid of eosinophils. Moreover, perivascular/peribronchial eosinophil numbers were reduced to levels indistinguishable from saline-challenged animals. This antibody-mediated depletion was not accompanied by effects on any other leukocyte population, including, but not limited to, T cells and mast cells/basophils. In addition, no effects were observed on other underlying allergic inflammatory responses in OVA-treated mice, including OVA-specific immunoglobulin production as well as T cell-dependent elaboration of Th2 cytokines. The ablation of virtually all pulmonary eosinophils in OVA-treated mice (i.e., without concurrent effects on T cell activities) resulted in a significant decrease in mucus accumulation and abolished allergen-induced airway hyperresponsiveness. These data demonstrate a direct causative relationship between allergen-mediated pulmonary pathologies and eosinophils.     

Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.     

ATP activates ataxia-telangiectasia mutated (ATM) in vitro. Importance of autophosphorylation

Ataxia-telangiectasia Mutated (ATM), mutated in the human disorder ataxia-telangiectasia, is rapidly activated by DNA double strand breaks. The mechanism of activation remains unresolved, and it is uncertain whether autophosphorylation contributes to activation. We describe an in vitro immunoprecipitation system demonstrating activation of ATM kinase from unirradiated extracts by preincubation with ATP. Activation is both time- and ATP concentration-dependent, other nucleotides fail to activate ATM, and DNA is not required. ATP activation is specific for ATM since it is not observed with kinase-dead ATM, it requires Mn2+, and it is inhibited by wortmannin. Exposure of activated ATM to phosphatase abrogates activity, and repeat cycles of ATP and phosphatase treatment reveal a requirement for autophosphorylation in the activation process. Phosphopeptide mapping revealed similarities between the patterns of autophosphorylation for irradiated and ATP-treated ATM. Caffeine inhibited ATM kinase activity for substrates but did not interfere with ATM autophosphorylation. ATP failed to activate either A-T and rad3-related protein (ATR) or DNA-dependent protein kinase under these conditions, supporting the specificity for ATM. These data demonstrate that ATP can specifically induce activation of ATM by a mechanism involving autophosphorylation. The relationship of this activation to DNA damage activation remains unclear but represents a useful model for understanding in vivo activation.