Vivo Interaction Research Articles

Chronic In Vivo Interaction of Dendritic Cells Expressing the Ligand Rae-1ε with NK Cells Impacts NKG2D Expression and Function.

To investigate how dendritic cells (DCs) interact with NK cells in vivo, we developed a novel mouse model in which Rae-1ε, a ligand of the NKG2D receptor, is expressed in cells with high levels of CD11c. In these CD11c-Rae1 mice, expression of Rae-1 was confirmed on all subsets of DCs and a small subset of B and T cells, but not on NK cells. DC numbers and activation status were unchanged, and NK cells in these CD11c-Rae1 mice presented the same Ly49 repertoire and maturation levels as their littermate wildtype controls. Early NK cell activation after mouse CMV infection was slightly lower than in wildtype mice, but NK cell expansion and viral control were comparable. Notably, we demonstrate that chronic interaction of NK cells with NKG2D ligand–expressing DCs leads to a reversible NKG2D down-modulation, as well as impaired NKG2D-dependent NK cell functions, including tumor rejection. In addition to generating a useful mouse model, our studies reveal in vivo the functional importance of the NK cell and DC cross-talk.

In Vivo Interaction Proteomics in Caenorhabditis elegans Embryos Provides New Insights into P Granule Dynamics

Studying protein interactions in whole organisms is fundamental to understanding development. Here, we combine in vivo expressed GFP-tagged proteins with quantitative proteomics to identify protein-protein interactions of selected key proteins involved in early C. elegans embryogenesis. Co-affinity purification of interaction partners for eight bait proteins resulted in a pilot in vivo interaction map of proteins with a focus on early development. Our network reflects known biology and is highly enriched in functionally relevant interactions. To demonstrate the utility of the map, we looked for new regulators of P granule dynamics and found that GEI-12, a novel binding partner of the DYRK family kinase MBK-2, is a key regulator of P granule formation and germline maintenance. Our data corroborate a recently proposed model in which the phosphorylation state of GEI-12 controls P granule dynamics. In addition, we find that GEI-12 also induces granule formation in mammalian cells, suggesting a common regulatory mechanism in worms and humans. Our results show that in vivo interaction proteomics provides unique insights into animal development.

Arabidopsis AtSerpin1, Crystal Structure and in Vivo Interaction with Its Target Protease RESPONSIVE TO DESICCATION-21 (RD21)

In animals, protease inhibitors of the serpin family are associated with many physiological processes, including blood coagulation and innate immunity. Serpins feature a reactive center loop (RCL), which displays a protease target sequence as a bait. RCL cleavage results in an irreversible, covalent serpin-protease complex. AtSerpin1 is an Arabidopsis protease inhibitor that is expressed ubiquitously throughout the plant. The x-ray crystal structure of recombinant AtSerpin1 in its native stressed conformation was determined at 2.2 A. The electrostatic surface potential below the RCL was found to be highly positive, whereas the breach region critical for RCL insertion is an unusually open structure. AtSerpin1 accumulates in plants as a full-length and a cleaved form. Fractionation of seedling extracts by nonreducing SDS-PAGE revealed the presence of an additional slower migrating complex that was absent when leaves were treated with the specific cysteine protease inhibitor L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane. Significantly, RESPONSIVE TO DESICCATION-21 (RD21) was the major protease labeled with the L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane derivative DCG-04 in wild type extracts but not in extracts of mutant plants constitutively overexpressing AtSerpin1, indicating competition. Fractionation by nonreducing SDS-PAGE followed by immunoblotting with RD21-specific antibody revealed that the protease accumulated both as a free enzyme and in a complex with AtSerpin1. Importantly, both RD21 and AtSerpin1 knock-out mutants lacked the serpin-protease complex. The results establish that the major Arabidopsis plant serpin interacts with RD21. This is the first report of the structure and in vivo interaction of a plant serpin with its target protease.

In Vivo Interaction of Steroid Receptor Coactivator (SRC)-1 and the Activation Function-2 Domain of the Thyroid Hormone Receptor (TR) β in TRβ E457A Knock-In and SRC-1 Knockout mice

The activation function-2 (AF-2) domain of the thyroid hormone (TH) receptor (TR)-beta is a TH-dependent binding site for nuclear coactivators (NCoA), which modulate TH-dependent gene transcription. In contrast, the putative AF-1 domain is a TH-independent region interacting with NCoA. We determined the specificity of the AF-2 domain and NCoA interaction by evaluating thyroid function in mice with combined disruption of the AF-2 domain in TRbeta, due to a point mutation (E457A), and deletion of one of the NCoAs, steroid receptor coactivator (SRC)-1. The E457A mutation was chosen because it abolishes NCoA recruitment in vitro while preserving normal TH binding and corepressor interactions resulting in resistance to TH. At baseline, disruption of SRC-1 in the homozygous knock-in (TRbeta(E457A/E457A)) mice worsened the degree of resistance to TH, resulting in increased serum T(4) and TSH. During TH deprivation, disruption of AF-2 and SRC-1 resulted in a TSH rise 50% of what was seen when AF-2 alone was removed, suggesting that SRC-1 was interacting outside of the AF-2 domain. Therefore, 1) during TH deprivation, SRC-1 is necessary for activating the hypothalamic-pituitary-thyroid axis; 2) ligand-dependent repression of TSH requires an intact AF-2; and 3) SRC-1 may interact with the another region of the TRbeta or the TRalpha to regulate TH action in the pituitary. This report demonstrates the dual interaction of NCoA in vivo: the TH-independent up-regulation possibly through another domain and TH-dependent down-regulation through the AF-2 domain.

Subcellular Localization and In Vivo Interactions of the Arabidopsis thaliana Ethylene Receptor Family Members

The gaseous phytohormone ethylene regulates many developmental processes and responses to environmental conditions in higher plants. In Arabidopsis thaliana, ethylene perception and initiation of signaling are mediated by a family of five receptors which are related to prokaryotic two-component sensor histidine kinases. The transient expression of fluorescence-tagged receptors in tobacco (Nicotiana benthamiana) epidermal leaf cells demonstrated that all ethylene receptors are targeted to the ER endomembrane network and do not localize to the plasmalemma. In support of in planta overlay studies, the ethylene receptors form homomeric and heteromeric protein complexes at the ER in living plant cells, as shown by membrane recruitment assays. A comparable in vivo interaction pattern was found in the yeast mating-based split-ubiquitin system. The overlapping but distinct expression pattern of the ethylene receptor genes suggests a differential composition of the ethylene receptor complexes in different plant tissues. Our findings may have crucial functional implications on the ethylene receptor-mediated efficiency of hormone perception, induction of signaling, signal attenuation and output.

Novel Murine Model To Study Modulation of Genes and Molecular Pathways Induced Following In Vivo Interaction between Multiple Myeloma Cells and Human BM Milieu.

Interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. This interaction within the bone marrow milieu is unique and its understanding is important in evaluating effects of novel agents in vitro and in vivo. We here describe a novel murine model that allows us to study the expression changes in vivo in MM cells within the human BM milieu. In this model, the green fluorescent protein (INA-6 GFP+) transduced IL-6-dependent human MM cell line, INA-6, was injected in human bone chip implanted into SCID mice. At different time points the bone chip was retrieved, cells flushed out and GFP+ MM cells were purified by CD138 MACS microbeads. Similar isolation process was used on INA-6 GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affymetrix) and DChip analyzer program. We have identified significant changes in expression of several genes following in vivo interaction between INA-6 and the BM microenvironment. Specifically, we observed up-regulation of genes associated with cytokines (IL-4, IL-8, IGFB 2–5) and chemokines (CCL2, 5, 6, 18, 24, CCR1, 2, 4), implicated in cell-cell signalling. Moreover genes implicated in DNA transcription (V-Fos, V-Jun, V-kit), adhesion (Integrin alpha 2b, 7, cadherin 1 and 11) and cell growth (CDC14, Cyclin G2, ADRA1A) were also up-regulated and genes involved in apoptosis and cell death (p-57, BCL2, TNF1a) were down-regulated. Using the Ingenuity Pathway Analysis the most relevant pathways modulated by the in vivo interaction between MM cells and BMSCs were IL-6, IGF1, TGF-beta and ERK/MAPK-mediated pathways as well as cell-cycle regulation and chemokine signalling. These results are consistent with previously observed in vitro cell signalling studies. Taken together these results highlight the ability of BM microenvironment to modulate the gene expression profile of the MM cells and our ability to in vivo monitor the changes. This model thus provides us with an ability to study in vivo effects of novel agents on expression profile of MM cells in BM milieu, to pre-clinically characterize their activity.

The C Terminus of the Immunophilin PASTICCINO1 Is Required for Plant Development and for Interaction with a NAC-like Transcription Factor

PASTICCINO1 (PAS1) is a high molecular weight FK506-binding protein (FKBP) involved in the control of cell proliferation and differentiation during plant development. Mutations in the C-terminal region of PAS1 result in severe developmental defects. We show here that the C-terminal domain of PAS1 controls the subcellular distribution of this protein. We also demonstrated in vitro and in vivo, by Forster resonance energy transfer, that this C-terminal region is required for interaction with FAN (FKBP-associated NAC), a new member of the plant-specific family of NAC transcription factors. PAS1 and FAN are translocated into the nucleus upon auxin treatment in plant seedlings. The nuclear translocation of PAS1 is dependent on the presence of the C terminus of the protein. Finally, we showed that FAN is involved in PAS1-regulated processes because FAN overproduction partly complemented the pas1 phenotype. We suggest that PAS1 regulates the function of this NAC-like transcription factor by controlling its targeting to the nucleus upon plant cell division.

Saccharomyces cerevisiae Lacking Btn1p Modulate Vacuolar ATPase Activity to Regulate pH Imbalance in the Vacuole

The vacuolar H(+)-ATPase (V-ATPase) along with ion channels and transporters maintains vacuolar pH. V-ATPase ATP hydrolysis is coupled with proton transport and establishes an electrochemical gradient between the cytosol and vacuolar lumen for coupled transport of metabolites. Btn1p, the yeast homolog to human CLN3 that is defective in Batten disease, localizes to the vacuole. We previously reported that Btn1p is required for vacuolar pH maintenance and ATP-dependent vacuolar arginine transport. We report that extracellular pH alters both V-ATPase activity and proton transport into the vacuole of wild-type Saccharomyces cerevisiae. V-ATPase activity is modulated through the assembly and disassembly of the V(0) and V(1) V-ATPase subunits located in the vacuolar membrane and on the cytosolic side of the vacuolar membrane, respectively. V-ATPase assembly is increased in yeast cells grown in high extracellular pH. In addition, at elevated extracellular pH, S. cerevisiae lacking BTN1 (btn1-Delta), have decreased V-ATPase activity while proton transport into the vacuole remains similar to that for wild type. Thus, coupling of V-ATPase activity and proton transport in btn1-Delta is altered. We show that down-regulation of V-ATPase activity compensates the vacuolar pH imbalance for btn1-Delta at early growth phases. We therefore propose that Btn1p is required for tight regulation of vacuolar pH to maintain the vacuolar luminal content and optimal activity of this organelle and that disruption in Btn1p function leads to a modulation of V-ATPase activity to maintain cellular pH homeostasis and vacuolar luminal content.

Gβγ Binds Histone Deacetylase 5 (HDAC5) and Inhibits Its Transcriptional Co-repression Activity

In a yeast two-hybrid screen designed to identify novel effectors of the G betagamma subunit of heterotrimeric G proteins, we found that G betagamma binds to histone deacetylase 5 (HDAC5), an enzyme involved in a pathway not previously recognized to be directly impacted by G proteins. Formation of the G beta1gamma2-HDAC5 complex in mammalian cells can be blocked by overexpression of G alpha(o), and this inhibition is relieved by activation of alpha2A-adrenergic receptor, suggesting that the interaction occurs in a signal-dependent manner. The C-terminal domain of HDAC5 binds directly to G betagamma through multiple motifs, and overexpression of this domain mimics the C terminus of G protein-coupled receptor kinase 2, a known G betagamma scavenger, in its ability to inhibit the G betagamma/HDAC5 interaction. The C terminus of HDAC4 shares significant similarity with that of HDAC5, and accordingly, HDAC4 is also able to form complexes with G beta1gamma2 in cultured cells, suggesting that the C-terminal domain of class II HDACs is a general G betagamma binding motif. Activation of a G(i/o)-coupled receptor results in a time-dependent activation of MEF2C, an HDAC5-regulated transcription factor, whereas inhibition of the interaction with a G betagamma scavenger inhibits MEF2C activity, suggesting a reduced potency of HDAC5-mediated inhibition. Taken together, these data imply that HDAC5 and possibly other class II HDACs can be added to the growing list of G betagamma effectors.

In Vivo Interaction between the Polyprenol Phosphate Mannose Synthase Ppm1 and the Integral Membrane Protein Ppm2 from Mycobacterium smegmatis Revealed by a Bacterial Two-hybrid System

Dolichol phosphate-mannose (Dol-P-Man) is a mannose donor in various eukaryotic glycosylation processes. So far, two groups of Dol-P-Man synthases have been characterized based on the way they are stabilized in the endoplasmic reticulum membrane. Enzymes belonging to the first group, such as the yeast Dpm1, are typical integral membrane proteins harboring a transmembrane segment (TMS) at their C terminus. In contrast, mammalian Dpm1, enzymes of the second group, lack the typical TMS and require the association with the small hydrophobic proteins Dpm3 to be properly stabilized in the endoplasmic reticulum membrane. In Mycobacterium tuberculosis, the Polyprenol-P-Man synthase MtPpm1 is involved in the biosynthesis of the cell wall-associated glycolipid lipoarabinomannan. MtPpm1 is composed of two domains. The C-terminal catalytic domain is homologous to eukaryotic Dol-P-Man synthases. The N-terminal domain of MtPpm1 contains six TMS that anchor the enzyme in the cytoplasmic membrane. In contrast, in Mycobacterium smegmatis, orthologs of the two domains of MtPpm1 are encoded by two distinct open reading frames, Msppm1 and Msppm2, organized as an operon. No TMS are predicted in MsPpm1, and subcellular fractionation experiments indicate that this enzyme is cytosolic when produced in Escherichia coli. Computer-assisted topology predictions and alkaline phosphatase insertions showed that MsPpm2 is an integral membrane protein. Using a recently developed bacterial two-hybrid system, it was found that MsPpm2 interacts with MsPpm1 to stabilize the synthase MsPpm1 in the bacterial membrane. This interaction is reminiscent of that of mammalian Dpm1 with Dpm3 and mimics the structure of MtPpm1 as demonstrated by the capacity of the two domains of MtPpm1 to spontaneously interact when co-expressed in E. coli.

Expression of a GRINL1A Protein in the Presence of an NMDA Receptor Subunit in Transfected Cells Reveals Co-Localization in the Plasma Membrane and Raises the Question of a Possible In Vivo Interaction

Expression of a GRINL1A Protein in the Presence of an NMDA Receptor Subunit in Transfected Cells Reveals Co-Localization in the Plasma Membrane and Raises the Question of a Possible In Vivo Interaction

In vivo interaction between NPR1 and transcription factor TGA2 leads to salicylic acid-mediated gene activation in Arabidopsis.

The Arabidopsis NPR1 protein is a key regulator of salicylic acid (SA)-mediated gene expression in systemic acquired resistance. Based on yeast two-hybrid analysis, NPR1 has been suggested to interact with members of the TGA family of transcription factors, including TGA2 (AHBP-1b). However, genetic evidence demonstrating that the NPR1-TGA interaction occurs in planta is still lacking, and the role of this interaction in SA-mediated gene activation has yet to be determined. In this study, we expressed a truncated form of TGA2 in Arabidopsis and found that the resulting transgenic lines displayed phenotypes similar to those of npr1 mutants. This dominant-negative effect of the TGA2 mutant shows that TGA2 and NPR1 interact in planta. We also present biochemical evidence indicating that this interaction is specific and enhanced by SA treatment. Moreover, using a chimera reporter system, we found that a chimeric TGA2GAL4 transcription factor activated a UAS(GAL)::GUS reporter gene in response to SA and that this activation was abolished in the npr1 mutant. NPR1 is required for the DNA binding activity of the transcription factor. These genetic data clearly demonstrate that TGA2 is a SA-responsive and NPR1-dependent transcription activator.

In Vivo Interaction between Chlorpyrifos and Parathion in Adult Rats: Sequence of Administration Can Markedly Influence Toxic Outcome

Organophosphorus insecticides (OPs) generally act through a common mechanism of toxicity initiated by inhibition of acetylcholinesterase (AChE). We studied the in vivo interactive toxicity of two common OPs, chlorpyrifos (CPF) and parathion (PS), in adult male rats. Dose–response studies estimated the acute oral LD1 values for the two OPs (CPF = 80 mg/kg po; PS = 4 mg/kg po) and these dosages or relative proportions were used to evaluate interactive toxicity. Three treatment strategies were evaluated: CPF followed by PS 4 h later (CPF-1st), PS followed by CPF 4 h later (PS-1st), and simultaneous (concurrent) exposures. Using LD1 dosages, rats in the CPF-1st and concurrent groups exhibited more cholinergic toxicity (i.e., salivation, lacrimation, urination, and diarrhea signs and involuntary movements) and higher lethality (7/8 and 6/8, respectively, beginning 1 h after PS) than those in the PS-1st group (2/8 lethality, beginning 3 days after CPF). Sequential exposures to lower dosages (CPF vs PS: 60 vs 3 mg/kg; 40 vs 2 mg/kg) led to more extensive neurotoxicity in the CPF-1st group compared to the other groups. Following lower dosages (40 vs 2 mg/kg), brain ChE inhibition was more extensive in the CPF-1st group at all time points (64–85%) and the concurrent group at 4 and 24 h after exposure (46–83%) compared to rats receiving PS first (7–48%). No differences were noted however, in plasma (71–93% inhibition) or liver (72–81%) cholinesterase activities nor were there group-related differences in plasma (50–60% inhibition) or liver (>85% inhibition) carboxylesterase activities. Incubation of liver samples with oxons in the presence or absence of calcium (i.e., 2 mM CaCl2 or EGTA) prior to addition of ChE (striatal sample) substantially blocked ChE inhibition by CPO (IC50: without liver = 4 nM; liver + calcium = 279 nM; liver + EGTA = 48 nM) but had lesser effects on PO-mediated inhibition (IC50: without liver = 17 nM; liver + EGTA = 56 nM; liver + calcium = 57 nM). Liver homogenate from animals preexposed to PS substantially decreased ChE inhibition by CPO when calcium was included (IC50: +EGTA = 8 nM; +calcium = 225 nM), but liver homogenate from animals preexposed to CPF was ineffective at blocking PO-induced inhibition (IC50: +EGTA = 16 nM; +calcium = 16 nM). We conclude that prior inhibition of carboxylesterase activity impacts toxicity of subsequent exposure to PS more than CPF because of more active detoxification of CPO by A-esterase. Together, these findings indicate that interactive toxicity from combined exposures to two OP insecticides can be markedly influenced by the sequence of administration.

In Vivo Interaction between Dynamitin and MacMARCKS Detected by the Fluorescent Resonance Energy Transfer Method

Dynamitin is a subunit of the dynactin complex regulating microtubule-dependent motor functions, and MacMARCKS (Macrophage-enriched myristoylated alanine-rich protein kinase C substrate) is a major protein kinase C substrate regulating integrin activation. The interaction between dynamitin and MacMARCKS has been implicated in integrin-dependent cell spreading. However, the in vivo interaction of these two proteins in living cells has not been demonstrated. Spatial and temporal information about the interaction is also lacking. In this study, we used the fluorescent resonance energy transfer method to demonstrate in vivo interaction between MacMARCKS and dynamitin with cyan fluorescent protein (CFP)-conjugated dynamitin as the donor fluorophore and yellow fluorescent protein (YFP)-conjugated MacMARCKS as the acceptor fluorophore. The interaction of these two fusion proteins was studied both in vitro and in vivo, and typical fluorescent resonance energy transfer was observed; the CFP emission peak increased while the YFP emission peak decreased when protein interaction was abolished. Spatial and temporal information was obtained in RAW macrophage cells. In resting macrophage cells, dynamitin-MacMARCKS interaction is concentrated at the cell periphery, although the majority of dynamitin is distributed at the perinuclear region of the cells. When cells were treated with phorbol 12-myristate 13-acetate, both proteins concentrated to perinuclear regions of the cells, and yet the interaction disappeared as the cell spread. Similar events were also observed in 293 cells. Thus, we conclude that dynamitin and MacMARCKS indeed interact in living cells.

In Vivo Interaction of the Adapter Protein CD2-associated Protein with the Type 2 Polycystic Kidney Disease Protein, Polycystin-2

We identified a developmentally regulated gene from mouse kidney whose expression is up-regulated in metanephrogenic mesenchyme cells when they are induced to differentiate to epithelial cells during kidney organogenesis. The deduced 70.5-kDa protein, originally named METS-1 (mesenchyme-to-epithelium transition protein with SH3 domains), has since been cloned as a CD2-associated protein (CD2AP). CD2AP is strongly expressed in glomerular podocytes, and the absence of CD2AP in mice results in congenital nephrotic syndrome. We have found that METS-1/CD2AP (hereafter referred to as CD2AP) is expressed at lower levels in renal tubular epithelial cells in the adult kidney, particularly in distal nephron segments. Independent yeast two-hybrid screens using the COOH-terminal region of either CD2AP or polycystin-2 as bait identified the COOH termini of polycystin-2 and CD2AP, respectively, as strong interacting partners. This interaction was confirmed in cultured cells by co-immunoprecipitation of endogenous polycystin-2 with endogenous CD2AP and vice versa. CD2AP shows a diffuse reticular cytoplasmic and perinuclear pattern of distribution, similar to polycystin-2, in cultured cells, and the two proteins co-localize by indirect double immunofluorescence microscopy. CD2AP is an adapter molecule that associates with a variety of membrane proteins to organize the cytoskeleton around a polarized site. Such a function fits well with that hypothesized for the polycystin proteins in renal tubular epithelial cells, and the present findings suggest that CD2AP has a role in polycystin-2 function.

Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors.

The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain-mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein-specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

The C-terminal (BRCT) Domains of BRCA1 Interact in Vivo with CtIP, a Protein Implicated in the CtBP Pathway of Transcriptional Repression

The BRCA1 tumor suppressor encodes a polypeptide with two recognizable protein motifs: a RING domain near the N terminus and two tandem BRCT domains at the C terminus. Studies of tumor-associated mutations indicate that the RING and BRCT sequences are required for BRCA1-mediated tumor suppression. In addition, recent work has shown that BRCA1 is a potent regulator of RNA transcription and that the BRCT domains are also essential for this activity. Therefore, we used the Sos recruitment system to screen for proteins that bind this critical region of BRCA1. Our results show that the BRCT domains interact in vivo with CtIP, a protein originally identified on the basis of its association with the CtBP transcriptional co-repressor. This finding suggests that BRCA1 regulates gene expression, at least in part, by modulating CtBP-mediated transcriptional repression. Moreover, the in vivo interaction between BRCA1 and CtIP is completely ablated by each of three independent tumor-associated mutations affecting the BRCT motifs of BRCA1. These results indicate that the BRCA1-CtIP interaction may be required for tumor suppression by BRCA1.

In Vivo Interaction of AR-L 115BS (Vardax®) with the Adrenergic Nervous System

In vitro studies have demonstrated that AR-L 115BS (AR-L), a new orally active nonglycosidic inotropic agent with vasodilator properties, does not act via adrenergic receptors. However, because AR-L is a phosphodiesterase inhibitor and interaction with the adrenergic nervous system may exist in vivo, we compared the actions of intravenous AR-L, isoproterenol, and propranolol, alone and in combination, in normal dogs. In seven awake morphine-sedated dogs, AR-L (4 mg/kg i.v.) did not alter circulating catecholamines despite increasing maximum rate of change of left ventricular pressure (dP/dt) by 76%. In anesthetized dogs, peak inotropic effect of AR-L occurred at 6 mg/kg (dP/dt from 5,450 +/- 1,280 to 12,000 +/- 3,050 mm Hg/s). Propranolol (1 mg/kg) depressed dP/dt from 5,725 +/- 2,032 to 2,530 +/- 631 mm Hg/s, and this was completely reversed by increasing doses of AR-L (2-30 mg/kg) but the maximum dP/dt attained in these dogs (6,050 +/- 221 mm Hg/s) remained below the level achieved by AR-L in the absence of propranolol. To determine if that difference was due to an interaction of AR-L with the adrenergic nervous system, the effect of AR-L on isoproterenol activity was studied in groups of beta-blocked and unblocked animals. In either group, the dose-response curve of dP/dt to isoproterenol was shifted upward by AR-L, but the actions of the two drugs were additive without real synergism (e.g., after propranolol:isoproterenol 10 micrograms/min 73%; AR-L 6 mg/kg 81%; both 160%. In unblocked dogs, the results were: isoproterenol 2 micrograms/min 96%; AR-L 1 mg/min 39%; both 138%). Similarly, isoproterenol and AR-L were only additive in their effects on heart rate and systemic vascular resistance. Thus, although AR-L is a phosphodiesterase inhibitor, its predominant mechanism of action appears to be independent of the adrenergic nervous system.

In Vivo Interaction of AR-L 115BS (Vardax®) with the Adrenergic Nervous System

Summary: In vitro studies have demonstrated that AR-L 115BS (AR-L), a new orally active nonglycosidic inotropic agent with vasodilator properties, does not act via adrenergic receptors. However, because AR-L is a phosphodiesterase inhibitor and interaction with the adrenergic nervous system may exist in vivo, we compared the actions of intravenous AR-L, isoproterenol, and propranolol, alone and in combination, in normal dogs. In seven awake morphine-sedated dogs, AR-L (4 mg/kg i.v.) did not alter circulating catecholamines despite increasing maximum rate of change of left ventricular pressure (dP/dt) by 76%. In anesthetized dogs, peak inotropic effect of AR-L occurred at 6 mg/kg (dP/dt from 5,450 ± 1,280 to 12,000 ± 3,050 mm Hg/s). Propranolol (1 mg/kg) depressed dP/dt from 5,725 ± 2,032 to 2,530 ± 631 mm Hg/s, and this was completely reversed by increasing doses of AR-L (2-30 mg/kg) but the maximum dP/dt attained in these dogs (6,050 ± 221 mm Hg/s) remained below the level achieved by AR-L in the absence of propranolol. To determine if that difference was due to an interaction of AR-L with the adrenergic nervous system, the effect of AR-L on isoproterenol activity was studied in groups of β-blocked and unblocked animals. In either group, the dose-response curve of dP/dt to isoproterenol was shifted upward by AR-L, but the actions of the two drugs were additive without real synergism (e.g., after propranolol: isoproterenol 10 μg/min 73%; AR-L 6 mg/kg 81%; both 160%. In unblocked dogs, the results were: isoproterenol 2 μg/min 96%; AR-L 1 mg/min 39%; both 138%). Similarly, isoproterenol and AR-L were only additive in their effects on heart rate and systemic vascular resistance. Thus, although AR-L is a phosphodiesterase inhibitor, its predominant mechanism of action appears to be independent of the adrenergic nervous system.

In Vivo interaction between RNA viruses isolated from the C57BL/Ka strain of mice

Virus replication can be initiated in the C57BL/Ka thymus by inoculation of three virus preparations isolated from mice of this strain, designated RadLV, RadLV-LTC, and BL/Ka(6), but not by three others, designated BL/Ka(B), BL/Ka(N), and BL/Ka(X). Coinfection by various mixtures of the three nonthymotropic viruses does not result in productive infection of the thymus. The addition of these nonthymotropic viruses to serial dilutions of RadLV, RadLV-LTC, or BL/Ka(6) does not significantly alter the replication of the wild-type RadLV preparations tested, whereas it significantly facilitates the replication of RadLV-LTC and BL/Ka(6). It is postulated that RadLV-LTC and BL/Ka(6) contain an attenuated or defective thymotropic and leukemogenic particle, the intrathymic replication of which is facilitated by coinfection with the nonthymotropic viruses.