Nonimmune Rabbit Serum Research Articles

Identification of In Vivo, Conserved, TAF15 RNA Binding Sites Reveals the Impact of TAF15 on the Neuronal Transcriptome

RNA binding proteins (RBPs) have emerged as major causative agents of amyotrophic lateral sclerosis (ALS). To investigate the function of TAF15, an RBP recently implicated in ALS, we explored its target RNA repertoire in normal human brain and mouse neurons. Coupling high-throughput sequencing of immunoprecipitated and crosslinked RNA with RNA sequencing and TAF15 knockdowns, we identified conserved TAF15 RNA targets and assessed the impact of TAF15 on the neuronal transcriptome. We describe a role of TAF15 in the regulation of splicing for a set of neuronal RNAs encoding proteins with essential roles in synaptic activities. We find that TAF15 is required for a critical alternative splicing event of the zeta-1 subunit of the glutamate N-methyl-D-aspartate receptor (Grin1) that controls the activity and trafficking of NR1. Our study uncovers neuronal RNA networks impacted by TAF15 and sets the stage for investigating the role of TAF15 in ALS pathogenesis.

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca2+ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca2+ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca2+ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca2+ that will enable it to act as a Ca2+ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca2+] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca2+ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca2+ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

Prognostic Significance of Stromal Platelet-Derived Growth Factor β-Receptor Expression in Human Breast Cancer

Prognostic Significance of Stromal Platelet-Derived Growth Factor β-Receptor Expression in Human Breast Cancer

Actin Pedestal Formation by Enteropathogenic Escherichia coli Is Regulated by IQGAP1, Calcium, and Calmodulin

During infection, enteropathogenic Escherichia coli (EPEC) injects effector proteins into the host cell to manipulate the actin cytoskeleton and promote formation of actin pedestals. IQGAP1 is a multidomain protein that participates in numerous cellular functions, including Rac1/Cdc42 and Ca(2+)/calmodulin signaling and actin polymerization. Here we report that IQGAP1, Ca(2+), and calmodulin modulate actin pedestal formation by EPEC. Infection with EPEC promotes both the interaction of IQGAP1 with calmodulin and the localization of IQGAP1 and calmodulin to actin pedestals while reducing the interaction of IQGAP1 with Rac1 and Cdc42. IQGAP1-null fibroblasts display a reduced polymerization of actin in response to EPEC. In addition, antagonism of calmodulin or chelation of intracellular Ca(2+) reduces EPEC-dependent actin polymerization. Furthermore, IQGAP1 specifically interacts with Tir in vitro and in cells. Together these data identify IQGAP1, Ca(2+), and calmodulin as a novel signaling complex regulating actin pedestal formation by EPEC.

Intracellular localization of “soluble” adenylate cyclase in potato plants

In animal cells, a key enzyme of the adenylate cuclase system, adenylate cyclase (AC), is represented by its transmembrane (tmAC) and soluble (sAC) forms [1]. Earlier, we have established that both forms of this enzyme are present in plants as well (potato), in nuclei and chloroplasts [2]. Using immunoprecepitation and Western blotting with antibody against animal sAC, we have found five sAC isoforms with mol wts of 225, 193, 92, 68, and 60 kD [3]. However, it is not clear until now, whether sAC is present only in nuclei and chloroplasts or it is localized also in other cell compartments, which could provide for more rapid transduction of external signals. Therefore, the objective of our work was to analyze intracellular sAC localization at the level of the cell ultrastructure. In this study, we applied for the first time the method of immuno-electronic cytochemistry for the studying of sAC intracellular localization in various potato tissues and its re-dislocation in the response to biotic stress. Tube-grown potato plantlets of two cultivars contrasting in their resistance to the causal agent of the ring rot were used in experiments: cv. Lugovskoi, resistant, and cv. Luk’yanovskii, susceptible. The root segments and leaf pieces from the upper part of the shoot of control plants (with roots not treated with extracellular polysaccharides (EPS) of the pathogen) and experimental plants kept for 2 h in the 0.1% EPS solution were fixed for 2 h in the mixture of 4% formaldehyde and 1% glutaraldehyde (Sigma, United States) prepared in 0.1 M Na-phosphate buffer, pH 7.2. Thereafter, samples were postfixed in 0.8% OsO 4 in the same buffer (2 h), dehydrated by the routine procedure, and embedded in the mixture of epoxide resins, Epon and Araldite. Immunocytochemistry was performed on ultrathin sections mounted on nickel grids, using primary rabbit polyclonal antibodies (PAB) against animal sAC at 1 : 200 dilution conjugated with 10-nm colloidal gold particles and secondary goat antibodies against rabbit immunoglobulins (SAB, Sigma, United States) at 1 : 30 dilution. All procedures were performed as described in [4], with the exception of the usage of the hoarse serum at 1 : 5 dilution as a blocking solution. Two control treatments were applied: (a) ultrathin sections not treated with PAB and (b) treatment with nonimmune rabbit serum instead PAB and then with SAB saturated with such serum. Specimens stained with uranyl acetate were examined with an LEO 906E electron microscope (Carl Zeiss, Germany), and the images were shown on the computer display. To evaluate sAC partition between cell compartments, we counted the number of gold particles in them. In each treatment, we analyzed 25 images; the total number of particles on the area of each compartment was calculated per 1 µ m 2 . The results were processed statistically. We observed the presence of SAB, reflecting sAC localization, in many intracellular compartments. Thus, in the cells of the root meristem and elongation zone, gold particles were seen in the cytosol, mitochondria,

CCR2 Receptor Is Essential to Activate Microbicidal Mechanisms to Control Toxoplasma gondii Infection in the Central Nervous System

Chemokines comprise a structurally related family of cytokines that regulate leukocyte trafficking. Because infection with Toxoplasma gondii can induce an important inflammatory reaction that, if left uncontrolled, can lead to death, we investigated the role of the chemokine receptor CCR2 in T. gondii infection. We orally infected CCR2(-/-) mice with five ME-49 T. gondii cysts and monitored morbidity, survival, and immune response thereafter. The CCR2(-/-) mice displayed higher susceptibility to infection as all mice died on day 28 after infection. Despite similar Th1 responses, a more evident anti-inflammatory response was induced in the peripheral organs of CCR2(-/-) mice compared with wild-type C57BL/6 mice. Additionally, CCR2(-/-) mice presented greater parasitism and a milder inflammatory reaction in their peripheral organs with lesser CD4(+) and MAC-1(+) and greater CD8(+) cell migration. The parasite load decreased in these organs in CCR2(-/-) mice but remained uncontrolled in the central nervous system. Additionally, we observed down-regulated inducible nitric oxide synthase expression in peripheral organs from CCR2(-/-) mice that was associated with a small nitric oxide production by spleen macrophages. In conclusion, in the absence of CCR2, another mechanism is activated to control tissue parasitism in peripheral organs. Nevertheless, CCR2 is essential for the activation of microbicidal mediators that control T. gondii replication in the central nervous system.

IQGAP1 Integrates Ca2+/Calmodulin and B-Raf Signaling

Ca(2+) and calmodulin modulate numerous cellular functions, ranging from muscle contraction to the cell cycle. Accumulating evidence indicates that Ca(2+) and calmodulin regulate the MAPK signaling pathway at multiple positions in the cascade, but the molecular mechanism underlying these observations is poorly defined. We previously documented that IQGAP1 is a scaffold in the MAPK cascade. IQGAP1 binds to and regulates the activities of ERK, MEK, and B-Raf. Here we demonstrate that IQGAP1 integrates Ca(2+) and calmodulin with B-Raf signaling. In vitro analysis reveals that Ca(2+) promotes the direct binding of IQGAP1 to B-Raf. This interaction is inhibited by calmodulin in a Ca(2+)-regulated manner. Epidermal growth factor (EGF) is unable to stimulate B-Raf activity in fibroblasts treated with the Ca(2+) ionophore A23187. In contrast, chelation of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) significantly enhances EGF-stimulated B-Raf activity, an effect that is dependent on IQGAP1. Incubation of cells with EGF augments the association of B-Raf with IQGAP1. Moreover, Ca(2+) regulates the association of B-Raf with IQGAP1 in cells. Increasing [Ca(2+)](i) with Ca(2+) ionophores significantly reduces co-immunoprecipitation of B-Raf and IQGAP1, whereas chelation of Ca(2+) enhances the interaction. Consistent with these findings, increasing and decreasing [Ca(2+)](i) increase and decrease, respectively, co-immunoprecipitation of calmodulin with IQGAP1. Collectively, our data identify a previously unrecognized mechanism in which the scaffold protein IQGAP1 couples Ca(2+) and calmodulin signaling to B-Raf function.

Serotonin Transamidates Rab4 and Facilitates Its Binding to the C Terminus of Serotonin Transporter

The serotonin transporter (SERT) on the plasma membrane is the major mechanism for the clearance of plasma serotonin (5-hydroxytryptamine (5HT)). The uptake rates of cells depend on the density of SERT molecules on the plasma membrane. Interestingly, the number of SERT molecules on the platelet surface is down-regulated when plasma 5HT ([5HT](ex)) is elevated. It is well reported that stimulation of cells with high [5HT](ex) induces transamidation of a small GTPase, Rab4. Modification with 5HT stabilizes Rab4 in its active, GTP-bound form, Rab4-GTP. Although investigating the mechanism by which elevated plasma 5HT level down-regulates the density of SERT molecules on the plasma membrane, we studied Rab4 and SERT in heterologous and platelet expression systems. Our data demonstrate that, in response to elevated [5HT](ex), Rab4-GTP co-localizes with and binds to SERT. The association of SERT with Rab4-GTP depends on: (i) 5HT modification and (ii) the GTP-binding ability of Rab4. Their association retains transporter molecules intracellularly. Furthermore, we mapped the Rab4-SERT association domain to amino acids 616-624 in the cytoplasmic tail of SERT. This finding provides an explanation for the role of the C terminus in the localization and trafficking of SERT via Rab4 in a plasma 5HT-dependent manner. Therefore, we propose that elevated [5HT](ex)"paralyzes" the translocation of SERT from intracellular locations to the plasma membrane by controlling transamidation and Rab4-GTP formation.

Protein Kinase Cϵ Mediates Polymeric Fibronectin Assembly on the Surface of Blood-borne Rat Breast Cancer Cells to Promote Pulmonary Metastasis

Malignant breast cancer cells that have entered the blood circulation from primary mammary fat pad tumors or are grown in end-over-end suspension culture assemble a characteristic, multi-globular polymeric fibronectin (polyFn) coat on their surfaces. Surface polyFn is critical for pulmonary metastasis, presumably by facilitating lung vascular arrest via endothelial dipeptidylpeptidase IV (CD26). Here, we show that cell-surface polyFn assembly is initiated by the state of suspension, is dependent upon the synthesis and secretion of cellular Fn, and is augmented in a dose- and time-dependent manner by plasma Fn. PolyFn assembly is regulated by protein kinase Cepsilon (PKCepsilon), which translocates rapidly and in increasing amounts from the cytosol to the plasma membrane and is phosphorylated. PolyFn assembly is impeded by select inhibitors of this kinase, i.e. bisindolylmaleimide I, Ro-32-0432, Gö6983, and Rottlerin, by the phorbol 12-myristate 13-acetate-mediated and time-dependent loss of PKCepsilon protein and decreased plasma membrane translocation, and more specifically, by stable transfection of lung-metastatic MTF7L breast cancer cells with small interfering RNA-PKCepsilon and dominant-negative PKCepsilon constructs (e.g. RD-PKCepsilon). The inability to assemble a cell surface-associated polyFn coat by knockdown of endogenous Fn or PKCepsilon impedes cancer cells from metastasis to the lungs. The present studies identify a novel regulatory mechanism for polyFn assembly on blood-borne breast cancer cells and depict its effect on pulmonary metastasis.

Disruption of the Insulin-like Growth Factor Type 1 Receptor in Osteoblasts Enhances Insulin Signaling and Action

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulin-like growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (DeltaIGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In DeltaIGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, DeltaIGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-(3)H]-deoxy-d-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of DeltaIGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.

Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice

Increased generation of reactive oxygen species (ROS) leads to oxidative stress in diabetes. Catalase is a highly conserved heme-containing protein that reduces hydrogen peroxide to water and oxygen and is an important factor decreasing cellular injury owing to oxidative stress. Hyperglycemic conditions increase oxidative stress and angiotensinogen gene expression. Angiotensinogen conversion to angiotensin II leads to a furtherance in oxidative stress through increased generation of reactive oxygen species. In this study, we utilized mice transgenically overexpressing rat catalase in a kidney-specific manner to determine the impact on ROS, angiotensinogen and apoptotic gene expression in proximal tubule cells of diabetic animals. Proximal tubules isolated from wild-type and transgenic animals without or with streptozotocin-induced diabetes were incubated in low glucose media in the absence or presence of angiotensin II or in a high-glucose media. Our results show that the overexpression of catalase prevents the stimulation of ROS and angiotensinogen mRNA in tubules owing to elevated glucose or angiotensin II in vitro. Additionally, overexpression of catalase attenuated ROS generation, angiotensinogen and proapoptotic gene expression and apoptosis in the kidneys of diabetic mice in vivo. Our studies point to an important role of ROS in the pathophysiology of diabetic nephropathy.

Pre-B-Cell Leukemia Transcription Factor 1 Regulates Expression of Valosin-Containing Protein, a Gene Involved in Cancer Growth

Valosin-containing protein (VCP) is involved in a wide variety of cellular functions. Our previous studies showed that the enhanced expression of VCP in cancer cells correlated with invasion and metastasis of cancers. Here, the regulatory mechanism for VCP transcription was investigated. Luciferase reporter constructs containing serially deleted 5'-flanking region of the VCP gene were transfected into MCF7 mammary carcinoma cell line, in which VCP was abundantly expressed. The deletion and mutation at the two binding motifs for pre-B-cell leukemia transcription factor 1 (PBX1) reduced the luciferase activity, indicating that these two PBX1 motifs mediated the transactivation of the VCP gene. Chromatin immunoprecipitation assay showed the binding of PBX-1 to the 5'-flanking region of the VCP gene. The knockdown of PBX1 by siRNA decreased the expression level of VCP. VCP is reported to maintain cell viability after the treatment of tumor necrosis factor-alpha. The viability of tumor necrosis factor-alpha-treated cells was significantly reduced in PBX1 knockdown MCF7. These findings indicate that PBX1 plays a crucial role in VCP expression and function and that the PBX-VCP pathway might be important for cell survival under cytokine stress.

Relaxin Enhances the Oncogenic Potential of Human Thyroid Carcinoma Cells

The role of members of the insulin-like superfamily in human thyroid carcinoma is primarily unknown. Here we demonstrate the presence of RLN2 relaxin and relaxin receptor LGR7 in human papillary, follicular, and undifferentiated anaplastic thyroid carcinoma suggesting a specific involvement of relaxin-LGR7 signaling in thyroid carcinoma. Stable transfectants of the LGR7-positive human follicular thyroid carcinoma cell lines FTC-133 and FTC-238 that secrete bioactive proRLN2 revealed this hormone to act as a multifunctional endocrine factor in thyroid carcinoma cells. Although RLN2 did not act as a mitogen, it acted as an autocrine/paracrine factor and significantly increased anchorage-independent growth and thyroid carcinoma cell motility and invasiveness through elastin matrices. Suppression of LGR7 expression by LGR7-siRNA abolished the RLN2-mediated accelerated tumor cell motility. The increased elastinolytic activity correlated with enhanced production and secretion of the lysosomal proteinases cathepsin-D (cath-D) and cath-L forms hereby identified as new RLN2 target molecules in human neoplastic thyrocytes. We found the intracellular distribution of procath-L specifically altered in RLN2 transfectants, providing first evidence for selective actions of relaxin on the powerful elastinolytic cath-L production, storage, and secretion in thyroid carcinoma cells. Thus, relaxin enhances the oncogenic potential and acts as novel endocrine modulator of invasiveness in human thyroid carcinoma cells.

The Novel WD-repeat Protein Morg1 Acts as a Molecular Scaffold for Hypoxia-inducible Factor Prolyl Hydroxylase 3 (PHD3)

Hypoxia-inducible factor-1 (HIF-1), a transcriptional complex composed of an oxygen-sensitive alpha- and a beta-subunit, plays a pivotal role in cellular adaptation to low oxygen availability. Under normoxia, the alpha-subunit of HIF-1 is hydroxylated by a family of prolyl hydroxylases (PHDs) and consequently targeted for proteasomal degradation. Three different PHDs have been identified, but the difference among their in vivo roles remain unclear. PHD3 is strikingly expressed by hypoxia, displays high substrate specificity, and has been identified in other signaling pathways. PHD3 may therefore hydroxylate divergent substrates and/or connect divergent cellular responses with HIF. We identified a novel WD-repeat protein, recently designated Morg1 (MAPK organizer 1), by screening a cDNA library with yeast two-hybrid assays. The interaction between PHD3 and Morg1 was confirmed in vitro and in vivo. We found seven WD-repeat domains by cloning the full-length cDNA of Morg1. By confocal microscopy both proteins co-localize within the cytoplasm and the nucleus and display a similar tissue expression pattern in Northern blots. Binding occurs at a conserved region predicted to the top surface of one propeller blade. Finally, HIF-mediated reporter gene activity is decreased by Morg1 and reduced to basal levels when Morg1 is co-expressed with PHD3. Suppression of Morg1 or PHD3 by stealth RNA leads to a marked increase of HIF-1 activity. These results indicate that Morg1 specifically interacts with PHD3 most likely by acting as a molecular scaffold. This interaction may provide a molecular framework between HIF regulation and other signaling pathways.

C5L2 Is a Functional Receptor for Acylation-stimulating Protein

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 +/- 33%, 5 microM ASP, p < 0.001, where basal = 100%) and glucose transport (168 +/- 21%, 10 microM ASP, p < 0.001). C3a similarly stimulates TGS (163 +/- 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 +/- 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 +/- 1.6, with ASP = 21.0 +/- 1.4 (144%), with ASP + oligonucleotides = 11.0 +/- 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 +/- 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 +/- 2.9, with ASP = 39.6 +/- 8.8 (177%), with ASP + oligonucleotides = 25.3 +/- 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged beta-arrestin, ASP induced beta-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.

Serum Amyloid A Binding to CLA-1 (CD36 and LIMPII Analogous-1) Mediates Serum Amyloid A Protein-induced Activation of ERK1/2 and p38 Mitogen-activated Protein Kinases

Serum amyloid A protein (SAA) is an acute-phase reactant, known to mediate pro-inflammatory cellular responses. This study reports that CLA-1 (CD36 and LIMPII Analogous-1; human orthologue of the Scavenger Receptor Class B Type I (SR-BI)) mediates SAA uptake and downstream SAA signaling. Flow cytometry experiments revealed more than a 5-fold increase of Alexa-488 SAA uptake in HeLa cells stably transfected with CLA-1. Alexa 488-HDL uptake directly correlated with SAA uptake when determined in several CLA-1 stably transfected HeLa cell clones expressing various levels of CLA-1. SAA directly binds to CLA-1 as determined by cross-linking and colocalization of anti-CLA-1 antibody with SAA. SAA was co-internalized with transferrin to the endocytic recycling compartment pointing to a potential site of SAA metabolism. Alexa-488 SAA uptake in the CLA-1-overexpressing HeLa cells, as well as in THP-1 monocyte cell line, can be efficiently blocked by unlabeled SAA, high density lipoprotein, and other CLA-1 ligands. At the same time, markedly enhanced levels of phosphorylation of the mitogen-activated protein kinases (MAPKs), ERK1/2, and p38, were observed in cells stably transfected with CLA-1 cells following SAA stimulation when compared with mock transfected cells. The levels of the SAA-induced interleukin-8 (IL-8) secretion by CLA-1-overexpressing cells also significantly exceeded (5- to 10-fold) those detected for control cells. Synthetic amphipathic peptides possessing a structural alpha-helical motif inhibited SAA-induced activation of both MAPKs and IL-8 secretion in THP-1 cells. The results of this study demonstrate for the first time that CLA-1 functions as an endocytic SAA receptor and is involved in SAA-mediated cell signaling events associated with the immune-related and inflammatory effects of SAA.

IgE-Dependent Activation of T cells by Allergen in Atopic Dermatitis: Pathophysiologic Relevance

The importance of interactions between allergen and IgE in allergen-mediated activation of T lymphocytes from patients with atopic dermatitis (AD) is unclear. A role for this interaction is implied by past evidence for IgE-facilitated presentation of allergen to T cells, but this phenomenon has only been demonstrated in specific in vitro systems biased to maximize the effect. It is therefore not known whether the process is relevant in patients. We now show that the responses to allergen of unmodified peripheral blood mononuclear cells (PBMC) from individual AD patients are significantly greater in the presence of fresh, unheated, IgE-containing autologous serum than the same serum heated under IgE-denaturing conditions or specifically depleted of IgE by immunoprecipitation. In six independent experiments, 59%-67% of the maximal in vitro PBMC response to allergen was found to be dependent upon the presence of IgE in autologous serum used at 5% final concentration. These data provide the first evidence that sufficient amounts of allergen-specific IgE and allergen-reactive T cells occur concomitantly in the blood of individual AD patients to allow IgE-enhanced T cell responses to allergen. We conclude that IgE-enhanced T cell responses are pathophysiologically relevant and a therapeutic target in AD.

Ureaplasma urealyticum binds mannose-binding lectin

Mannose-binding C-type lectin (MBL) is an important component of innate immunity in mammals. Mannose-binding lectin (MBL), an acute phase protein, acts as an opsonin for phagocytosis and also activates the mannan-binding lectin complement pathway. It may play a particularly significant role during infancy before adequate specific protection can be provided by the adaptive immune system. Ureaplasma urealyticum has been linked to several diseases including pneumonia and chronic lung disease (CLD) in premature infants. We therefore investigated the ability of U. urealyticum to bind MBL. A guinea pig IgG anti-rabbit-MBL antiserum was produced. An immunoblot (dot-blot) assay done on nitrocellulose membrane determined that the anti-MBL antibody had specificity against both rabbit and human MBL. Pure cultures of U. urealyticum, serotype 3, were used to make slide preparations. The slides containing the organisms were then incubated with nonimmune rabbit serum containing MBL. Ureaplasma was shown to bind rabbit MBL with an immunocytochemical assay using the guinea pig IgG anti-rabbit MBL antiserum. Horseradish peroxidase (HRP)-labeled anti-guinea pig IgG was used to localize the reaction. The anti-MBL antiserum was also used in an immunocytochemical assay to localize U. urealyticum in histological sections of lungs from mice specifically infected with this organism. The same method also indicated binding of MBL by ureaplasma in human lung tissue obtained at autopsy from culture positive infants. Our results demonstrate that ureaplasma has the capacity to bind MBL. The absence of MBL may play a role in the predisposition of diseases related to this organism.